Vaccine and method for preventing biofilm formation

ABSTRACT

The present invention is directed to compounds and methods for immunizing a patient against a biofilm-producing bacterial infection and a vaccine related thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/438,600, filed Jan. 7, 2003, and U.S. Provisional Application No.60/502,303, filed Sep. 12, 2003, which applications are incorporatedherein by reference.

U.S. GOVERNMENT RIGHTS

[0002] This work was supported by the United States Public HealthService NIAID, National Institutes of Health, Grants AI24616 andAI30040. The United States Government may have certain rights to thisinvention.

BACKGROUND OF THE INVENTION

[0003] Over the past ten years, studies have shown that biofilmformation by bacteria is a factor in their ability to survive onartificial and mucosal surfaces. Recent studies have indicated thatnontypeable Haemophilus influenzae (NTHi) produces biofilms duringmiddle ear infection in animal models. Bacteria growing as a biofilmdisplay a different phenotype than free-living bacteria. They havegreatly reduced metabolic rates that render them nearly impervious toantimicrobial treatment, and they have an exopolysaccharide matrix thatprovides protection from phagocytosis and other host defense mechanisms.They also demonstrate reliance on complex intercellular communicationsystems that provide for organized growth characteristics. Further, theyare recalcitrant to standard culture techniques because of their alteredmetabolism.

[0004] The reduced metabolic and divisional rates of biofilm bacterialargely explain the failure of antibiotics to eliminate infections inpatients who have biofilm-colonized indwelling medical devices,primarily because non-dividing bacteria largely escape antibiotickilling. Antibiotic treatment of biofilms kills bacteria on theperiphery, but deep organisms persist and act as a nidus for regrowthand periodic planktonic (i.e.,“free-floating” bacterial) showers, thatcan result in systemic infection.

[0005] There exists a need in the art for a method to treatinginfections associated with biofilm-producing bacteria.

SUMMARY OF THE INVENTION

[0006] Recent evidence indicates that biofilm formation occurs in otitismedia (OM) and is an important mechanism by which nontypeableHaemophilus influenzae (NTHi) causes this disease. As described herein,it has been discovered that H. influenzae 2019 lsgG and 2019 rfe areinvolved in NTHi biofilm formation, and that mutation of these genesprevents biofilm formation. The induction of antibodies by NTHi LsgG andRfe vaccines inactivate these proteins and prevent biofilm formation.

[0007] The present invention provides a vaccine comprising animmunogenic amount of a biofilm peptide, which amount is effective toimmunize a patient against a biofilm-producing bacterial infection, forexample, a Haemophilus influenzae infection, in combination with aphysiologically-acceptable, non-toxic vehicle. In one embodiment of theinvention, the vaccine comprises a biofilm peptide from a Haemophilusinfluenzae. As used herein, the term “biofilm peptide” includes variantsor biologically active or inactive fragments of this polypeptide. A“variant” of the polypeptide is a biofilm peptide that is not completelyidentical to a native biofilm peptide. A variant biofilm peptide can beobtained by altering the amino acid sequence by insertion, deletion orsubstitution of one or more amino acid. The amino acid sequence of theprotein is modified, for example by substitution, to create apolypeptide having substantially the same or improved qualities ascompared to the native polypeptide. In one embodiment, the biofilmpeptide is a LsgG gene product and/or a Rfe gene product. The infectioncan be a chronic infection. The infection can be caused by abacterial-biofilm. The infection can be otitis media (OM), otitis mediawith effusion (OME), or chronic bronchitis.

[0008] The present invention also provides a method of treating orpreventing a Haemophilus influenzae infection, comprising administeringto a patient such a vaccine.

[0009] The present invention additionally provides a method ofpreventing infection or colonization of Haemophilus influenzae in apatient by administering to the patient an agent that inhibits theproduction of a Haemophilus influenzae biofilm peptide.

[0010] In addition, the present invention provides an isolated andpurified Haemophilus influenzae cell comprising a disrupted biofilmgene, for example, LsgG or Rfe, of the cell, wherein the disruptionresults in a reduction of biofilm formation in the transgenicHaemophilus influenzae cell as compared to a wild-type Haemophilusinfluenzae cell. In one embodiment, the biofilm gene can is disrupted byinsertional inactivation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 depicts the LsgG deletion-replacement used to transformNTHi 2019 to generate a chromosomal lsgG deletion.

[0012]FIG. 2 depicts Haemophilus influenzae LsgG amino acid sequence(SEQ ID NO:1), the assigned homology of which is molybdenum regulatoryprotein (modE), and Haemophilus influenzae LsgG nucleic acid sequence(SEQ ID NO:2).

[0013]FIG. 3 depicts the rfe deletion-replacement used to transform NTHi2019 to generate a chromosomal rfe deletion.

[0014]FIG. 4 depicts Haemophilus influenzae Rfe amino acid sequence (SEQID NO:3), the assigned homology of which is undecaprenyl-phosphatealpha-N-acetylglucosaminyltransferase (rfe), and Haemophilus influenzaeRfe nucleic acid sequence (SEQ ID NO:4).

[0015]FIG. 5. O'Toole-Kolter assay showing evidence of biofilm formationby NTHi and the effects on biofilm formation by lsgA, rfe and IsgGmutations. Lane 1, 2, and 3 are NTHi strain 2019, 3198, 7502, lane 4contains NTHi2019 lsgA, lane 6 contains NTHi2019 lsgG and lane 7contains NTHI2019rfe. The results are presented as the mean of fourindependent experiments and the error bars equal one standard deviation.The values obtain with the lsgG and rfe were statistically highlysignificant (p<0.005) when compared to biofilm produced by NTHi 2019using paired T-test analysis.

[0016]FIG. 6. Continuous flow chamber for the analysis of biofilmformation by NTHi. Panel A shows a schematic representation of the flowchamber. The top surface is a glass cover slip (dotted lines) to whichthe biofilm adheres. This is sealed to the chamber with silicone cement.The chamber is infected with either 10⁷ NTHi 2019 gfp, NTHi 2019lsgG gfpor NTHi 2019rfe gfp. A continuous flow of defined medium essentialmedium diluted 1:10 is flowed through the chamber at 180 microliters perminute. The development biofilms is monitored by confocal microscopyevery 24 hours for 4 to seven days. Panel B shows a chamber infectedwith NTHi 2019.

[0017]FIG. 7 shows the confocal examination of flow chamber studies ofstrain NTHi 2019::gfp at day one (7A and 7B). FIG. 7B represents avertical cross-section of the Z-series shown in FIG. 7A. As can be seen,a biofilm with a vertical height of approximately 15 microns forms overa 4 day NTHi 2019 infection while minimal to no biofilm forms during asimilar period of infection with strains NTHi 2019rfe::gfp (FIG. 11A and11B) or NTHi 2019lsgG::gfp (FIG. 12A and 12B).

[0018]FIG. 8 shows the confocal examination of flow chamber studies ofstrain NTHi 2019rfe::gfp at day one (8A and 8B). FIG. 8B represents avertical cross-section of the Z-series shown in FIG. 8A.

[0019]FIG. 9 shows the confocal examination of flow chamber studies ofstrain NTHi 2019lsgG::gfp at day one (9A and 9B). FIG. 9B represents avertical cross-section of the Z-series shown in FIG. 9A.

[0020]FIG. 10 shows the confocal examination of flow chamber studies ofstrain NTHi 2019::gfp at day four (10A and 10B). FIG. 10B represents avertical cross-section of the Z-series shown in FIG. 10A.

[0021]FIG. 11 shows the confocal examination of flow chamber studies ofstrain NTHi 2019rfe::gfp at day four (11A and 11B). FIG. 11B representsa vertical cross-section of the Z-series shown in FIG. 11A.

[0022]FIG. 12 shows the confocal examination of flow chamber studies ofstrain NTHi 2019lsgG::gfp at day four (12A and 12B). FIG. 12B representsa vertical cross-section of the Z-series shown in FIG. 12A.

[0023]FIG. 13 shows confocal images of primary bronchial epithelialcells infected for four days with NTHI 2019::gfp (13A), NTHi2019rfe::gfp (13B) and NTHi 2019lsgG::gfp (13C). As can be seen, denseNTHi 2019 biofilm patches covers the epithelial cell surface while onlyisolated clusters of NTHi 2019rfe::gfp and NTHi 2019lsgG::gfp.

[0024]FIG. 14 shows biofilm formation by NTHi strain 2019 after fivedays of growth in a continuous flow chamber (14A) and a toluidineblue-stained cryosection of the NTHi day five biofilm embedded in OCT(14B). The bottom of the section is adjacent to the glass coverslipsurface.

[0025]FIG. 15 shows scanning electron micrographs of a five-day biofilm.FIG. 15A is a view of the top surface of the biofilm. The surface hascracked during the desiccation and dehydration processes, but the matrixof the biofilm surrounding organisms can be seen. FIG. 15B shows across-sectional view of the biofilm. The coverslip surface upon whichthe biofilm formed is at the top. Multiple water channels can be seenthroughout the biofilm. FIG. 15C shows a higher magnification of thecross-sectional view. Fibrillous material can be seen connecting theorganisms within the biofilm. The scale bars are given on each image.

[0026]FIG. 16 shows the results of confocal microscopy of live/deadstaining of the biofilm at day 2 (A) and day 5 (B). The panels arepresented as vertical sections of a Z-series comprised of 10-five micronoptical sections. The biofilms are embedded in OCT and werecryosectioned. Viable organisms can be seen in each specimen; however,by five days a greater proportion of the organisms at the coverslipsurface appear to be non-viable, based on this assay. The scale barsrepresent 100 μm.

[0027]FIG. 17. Mass spectrometry analysis of NTHi 2019 LOS isolated fromplate-grown, day five biofilm, and day five planktonic organisms.Sialylated glycoforms are designated in red. A completed description ofthe spectra is given in the text and Tables 2 and 3.

[0028]FIG. 18. Results of O'Toole-Kolter assays of strain 2019 and eight2019 mutants are shown in FIG. 18. As can be seen, three mutants, siaA,siaB, and wecA, produced significantly less biofilm than the parentstrain. The other mutants produced biofilm in amounts similar to orgreater than the parent strain.

[0029]FIG. 19 shows vertical cross-sections comprised of 60 one micronoptical sections of confocal microscopic studies of day five continuousflow chambers infected with NTHi 2019 gfp (panelA), NTHi 2019 wecA.:gfp(panel B), NTHi 2019 siaB::gfp (panel C), NTHi 2019 gfp in mediumwithout NeuAc supplementation (panel E) and NTHi 2019 gfp in mediumsupplemented with 20 μg/ml NeuAc supplementation (panel D).

[0030]FIG. 20. O'Toole-Kolter assays demonstrating the reduction ofbiofilm when NTHi 2019 is grown in defined medium with

and without NeuAc

supplementation is seen in FIG. 20A. Each results is mean result from ofeight wells. The bars represent one standard deviation. The differencein biofilm between growth in 20 μM NeuAc and NeuAc-free media issignificant (p<0.005). FIG. 20B shows the results of NeuAc incorporationafter 24 hour growth of NTHi 2019 nanA

and NTHi 2019 nanA::pgm

in defined medium in the presence of ¹⁴C NeuAc. Each results representsthe results of studies from nine individual wells. The error barsrepresent 1 standard deviation. This study demonstrates that NTHi 2019nanA::pgm is incorporating NeuAc into a carbohydrate structure. Sincethis mutant cannot incorporate NeuAc into its LOS, the NeuAc is mostlikely being incorporated into biofilm. The uptake in six control wellsto which bacteria were not added is also shown

[0031]FIG. 21 shows a confocal micrograph of OCT-embedded cryosectionedbiofilm stained with Sambucus nigrans-TRITC (panel A) and Maachiaamurensis-FITC (panel B). The merged image of panel A and B is shown inpanel C.

[0032]FIG. 22 shows a confocal micrograph of OCT-embedded cryosectionedbiofilm stained with Sambucus nigrans-TRITC (panel A) and Maachiaamurensis-FITC (panel B) after 1 hour exposure to 0.05 units of Vibriocholera neuraminidase. The merged image of panels A and B is shown inpanel C. The scale bars represent 50 μm.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0033] A “biofilm” is a complex organization of bacteria that areanchored to a surface via a bacterially extruded exopolysaccharidematrix, and grow into differentiated towers that can be several hundredbacteria in height (Costerton et al., 1999). The extrudedexopolysaccharide matrix, which comprises more than 90% of the biofilm,envelopes the bacteria and provides protection from phagocytosis andoxidative burst mechanisms, both in natural environments and in thehost. Bacteria within biofilms are also resistant to the host's humoraldefense systems because or a lack of accessibility by immunoglobulin andcomplement. The attachment of bacteria to a surface triggers theexpression of a cassette of genes, which results in the formation of abiofilm. A “biofilm phenotype” confers to a bacterium a reducedmetabolic activity and enhanced antibiotic resistance in comparison withthe corresponding planktonic phenotype. A “biofilm-producing bacterium”or “biofilm bacterium” is a bacterium capable of producing, forming,and/or accumulating a biofilm in vitro or in vivo, e.g., on artificialand mucosal surfaces. Biofilm-producing bacteria include, but are notlimited to, Haemophilus influenzae. Biofilm bacteria have beendemonstrated to be highly resistant to growth in standard planktonicculture, attributed to differences in gene expression.

[0034] As used herein, “disrupted gene” refers to an insertion,substitution, or deletion either in a gene of interest or in thevicinity of the gene, i.e., upstream (5′) or downstream (3′) of thegene, which results in the reduction of the biological activity or theloss of substantially all of the biological activity associated with thegene's product. For example, a disrupted gene involved in biofilmproduction and/or formation (a “biofilm-gene”), e.g., LsgG or Rfe, wouldbe unable to express a protein related to the production or formation ofa biofilm (a “biofilm protein” or a “biofilm peptide”), e.g., a proteinassociated with molybdate uptake and incorporation, fumerate metabolism,iron utilization, carbohydrate biosynthesis and cross-linking, andanaerobic respiration. A gene can be disrupted by any one of a number ofmethods known to the art.

[0035] The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucl. Acids Res., 19:508 (1991);Ohtsuka et al., JBC, 260:2605 (1985); Rossolini et al., Mol. Cell.Probes, 8:91 (1994). A “nucleic acid fragment” is a fraction of a givennucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority oforganisms is the genetic material while ribonucleic acid (RNA) isinvolved in the transfer of information contained within DNA intoproteins. The term “nucleotide sequence” refers to a polymer of DNA orRNA that can be single-or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The terms “nucleic acid”,“nucleic acid molecule”, “nucleic acid fragment”, “nucleic acid sequenceor segment”, or “polynucleotide” may also be used interchangeably withgene, cDNA, DNA and RNA encoded by a gene.

[0036] The invention encompasses isolated or substantially purifiednucleic acid or protein compositions. In the context of the presentinvention, an “isolated” or “purified” DNA molecule or an “isolated” or“purified” polypeptide is a DNA molecule or polypeptide that existsapart from its native environment and is therefore not a product ofnature. An isolated DNA molecule or polypeptide may exist in a purifiedform or may exist in a non-native environment such as, for example, atransgenic host cell. For example, an “isolated” or “purified” nucleicacid molecule or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In oneembodiment, an “isolated” nucleic acid is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated nucleic acid molecule can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences thatnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived. A protein that is substantiallyfree of cellular material includes preparations of protein orpolypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) ofcontaminating protein. When the protein of the invention, orbiologically active portion thereof, is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of- interestchemicals. Fragments and variants of the disclosed nucleotide sequencesand proteins or partial-length proteins encoded thereby are alsoencompassed by the present invention. By “fragment” or “portion” ismeant a full length or less than full length of the nucleotide sequenceencoding, or the amino acid sequence of, a polypeptide or protein.

[0037] The term “gene” is used broadly to refer to any segment ofnucleic acid associated with a biological function. Thus, genes includecoding sequences and/or the regulatory sequences required for theirexpression. For example, gene refers to a nucleic acid fragment thatexpresses mRNA, functional RNA, or specific protein, includingregulatory sequences. Genes also include nonexpressed DNA segments that,for example, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

[0038] “Naturally occurring” is used to describe an object that can befound in nature as distinct from being artificially produced. Forexample, a protein or nucleotide sequence present in an organism(including a virus), which can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory, isnaturally occurring.

[0039] The term “chimeric” refers to any gene or DNA that contains 1)DNA sequences, including regulatory and coding sequences, that are notfound together in nature, or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may comprise regulatory sequencesand coding sequences that are derived from different sources, orcomprise regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different from that found in nature.

[0040] A “transgene” refers to a gene that has been introduced into thegenome by transformation and is stably maintained. Transgenes mayinclude, for example, DNA that is either heterologous or homologous tothe DNA of a particular cell to be transformed. Additionally, transgenesmay comprise native genes inserted into a non-native organism, orchimeric genes. The term “endogenous gene” refers to a native gene inits natural location in the genome of an organism. A “foreign” generefers to a gene not normally found in the host organism but that isintroduced by gene transfer.

[0041] The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

[0042] A “variant” of a molecule is a sequence that is substantiallysimilar to the sequence of the native molecule. For nucleotidesequences, variants include those sequences that, because of thedegeneracy of the genetic code, encode the identical amino acid sequenceof the native protein. Naturally occurring allelic variants such asthese can be identified with the use of well-known molecular biologytechniques, as, for example, with polymerase chain reaction (PCR) andhybridization techniques. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis that encode the nativeprotein, as well as those that encode a polypeptide having amino acidsubstitutions. Generally, nucleotide sequence variants of the inventionwill have at least 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%,at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, to 98%, sequence identity to the native (endogenous)nucleotide sequence.

[0043] “Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

[0044] “Recombinant DNA molecule” is a combination of DNA sequences thatare joined together using recombinant DNA technology and procedures usedto join together DNA sequences as described, for example, in Sambrookand Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (3^(rd) edition, 2001).

[0045] The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” each refer to a sequence that originatesfrom a source foreign to the particular host cell or, if from the samesource, is modified from its original form. Thus, a heterologous gene ina host cell includes a gene that is endogenous to the particular hostcell but has been modified. The terms also include non-naturallyoccurring multiple copies of a naturally occurring DNA sequence. Thus,the terms refer to a DNA segment that is foreign or heterologous to thecell, or homologous to the cell but in a position within the host cellnucleic acid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides.

[0046] A “homologous” DNA sequence is a DNA sequence that is naturallyassociated with a host cell into which it is introduced.

[0047] “Wild-type” refers to the normal gene, or organism found innature without any known mutation.

[0048] “Genome” refers to the complete genetic material of an organism.

[0049] A “vector” is defined to include, inter alia, any plasmid,cosmid, phage or binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.,autonomous replicating plasmid with an origin of replication).

[0050] “Cloning vectors” typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss ofessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance, hygromycin resistance orampicillin resistance.

[0051] “Expression cassette” as used herein means a DNA sequence capableof directing expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one that isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter that initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development.

[0052] Such expression cassettes will comprise the transcriptionalinitiation region of the invention linked to a nucleotide sequence ofinterest. Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the gene of interest to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

[0053] “Coding sequence” refers to a DNA or RNA sequence that codes fora specific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

[0054] The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

[0055] A “functional RNA” refers to an antisense RNA, ribozyme, or otherRNA that is not translated.

[0056] The term “RNA transcript” refers to the product resulting fromRNA polymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

[0057] “Regulatory sequences” and “suitable regulatory sequences” eachrefer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences include enhancers, promoters, translation leader sequences,introns, and polyadenylation signal sequences. They include natural andsynthetic sequences as well as sequences that may be a combination ofsynthetic and natural sequences. As is noted above, the term “suitableregulatory sequences” is not limited to promoters. However, somesuitable regulatory sequences useful in the present invention willinclude, but are not limited to constitutive promoters, tissue-specificpromoters, development-specific promoters, inducible promoters and viralpromoters.

[0058] “5′ non-coding sequence” refers to a nucleotide sequence located5′ (upstream) to the coding sequence. It is present in the fullyprocessed mRNA upstream of the initiation codon and may affectprocessing of the primary transcript to mRNA, mRNA stability ortranslation efficiency (Turner et al., Mol. Biotech., 3:225 (1995).

[0059] “3′ non-coding sequence” refers to nucleotide sequences located3′ (downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

[0060] The term “translation leader sequence” refers to that DNAsequence portion of a gene between the promoter and coding sequence thatis transcribed into RNA and is present in the fully processed mRNAupstream (5′) of the translation start codon. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency.

[0061] The term “mature” protein refers to a post-translationallyprocessed polypeptide without its signal peptide. “Precursor” proteinrefers to the primary product of translation of an mRNA. “Signalpeptide” refers to the amino terminal extension of a polypeptide, whichis translated in conjunction with the polypeptide forming a precursorpeptide and which is required for its entrance into the secretorypathway. The term “signal sequence” refers to a nucleotide sequence thatencodes the signal peptide.

[0062] “Promoter” refers to a nucleotide sequence, usually upstream (5′)to its coding sequence, which controls the expression of the codingsequence by providing the recognition for RNA polymerase and otherfactors required for proper transcription. “Promoter” includes a minimalpromoter that is a short DNA sequence comprised of a TATA- box and othersequences that serve to specify the site of transcription initiation, towhich regulatory elements are added for control of expression.“Promoter” also refers to a nucleotide sequence that includes a minimalpromoter plus regulatory elements that is capable of controlling theexpression of a coding sequence or functional RNA. This type of promotersequence consists of proximal and more distal upstream elements, thelatter elements often referred to as enhancers. Accordingly, an“enhancer” is a DNA sequence that can stimulate promoter activity andmay be an innate element of the promoter or a heterologous elementinserted to enhance the level or tissue specificity of a promoter.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even be comprised of synthetic DNA segments. A promoter mayalso contain DNA sequences that are involved in the binding of proteinfactors that control the effectiveness of transcription initiation inresponse to physiological or developmental conditions.

[0063] The “initiation site” is the position surrounding the firstnucleotide that is part of the transcribed sequence, which is alsodefined as position +1. With respect to this site all other sequences ofthe gene and its controlling regions are numbered. Downstream sequences(i.e. further protein encoding sequences in the 3′ direction) aredenominated positive, while upstream sequences (mostly of thecontrolling regions in the 5′ direction) are denominated negative.

[0064] Promoter elements, particularly a TATA element, that are inactiveor that have greatly reduced promoter activity in the absence ofupstream activation are referred to as “minimal or core promoters.” Inthe presence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

[0065] “Constitutive expression” refers to expression using aconstitutive or regulated promoter. “Conditional” and “regulatedexpression” refer to expression controlled by a regulated promoter.

[0066] “Operably-linked” refers to the association of nucleic acidsequences on single nucleic acid fragment so that the function of one isaffected by the other. For example, a regulatory DNA sequence is said tobe “operably linked to” or “associated with” a DNA sequence that codesfor an RNA or a polypeptide if the two sequences are situated such thatthe regulatory DNA sequence affects expression of the coding DNAsequence (i.e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

[0067] “Expression” refers to the transcription and/or translation in acell of an endogenous gene, transgene, as well as the transcription andstable accumulation of sense (mRNA) or functional RNA. In the case ofantisense constructs, expression may refer to the transcription of theantisense DNA only. Expression may also refer to the production ofprotein.

[0068] “Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples oftranscription stop fragments are known to the art.

[0069] “Translation stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as one or more terminationcodons in all three frames, capable of terminating translation.Insertion of a translation stop fragment adjacent to or near theinitiation codon at the 5′ end of the coding sequence will result in notranslation or improper translation. Excision of the translation stopfragment by site-specific recombination will leave a site-specificsequence in the coding sequence that does not interfere with propertranslation using the initiation codon.

[0070] The terms “cis-acting sequence” and “cis-acting element” refer toDNA or RNA sequences whose functions require them to be on the samemolecule.

[0071] The terms “trans-acting sequence” and “trans-acting element”refer to DNA or RNA sequences whose function does not require them to beon the same molecule.

[0072] “Chromosomally-integrated” refers to the integration of a foreigngene or DNA construct into the host DNA by covalent bonds. Where genesare not “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

[0073] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence,” (b) “comparison window,” (c) “sequence identity,”(d) “percentage of sequence identity,” and (e) “substantial identity.”

[0074] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0075] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

[0076] Methods of alignment of sequences for comparison are well knownin the art. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, CABIOS, 4:11 (1988); the local homology algorithmof Smith et al., Adv. Appl. Math., 2:482 (1981); the homology alignmentalgorithm of Needleman and Wunsch, JMB, 48:443 (1970); thesearch-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad.Sci. USA, 85:2444 (1988); the algorithm of Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 87:2264 (1990), modified as in Karlin andAltschul, Proc. Natl. Acad. Sci. USA, 90:5873 (1993).

[0077] Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View,California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST,FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8(available from Genetics Computer Group (GCG), 575 Science Drive,Madison, Wis., USA). Alignments using these programs can be performedusing the default parameters. The CLUSTAL program is well described byHiggins et al., Gene, 73:237 (1988); Higgins et al., CABIOS, 5:151(1989); Corpet et al., Nucl. Acids Res., 16:10881 (1988); Huang et al.,CABIOS, 8:155 (1992); and Pearson et al., Meth. Mol. Biol., 24:307(1994). The ALIGN program is based on the algorithm of Myers and Miller,supra. The BLAST programs of Altschul et al., JMB, 215:403 (1990); Nucl.Acids Res., 25:3389 (1990), are based on the algorithm of Karlin andAltschul supra.

[0078] Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information (available onthe world wide web at ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always>0) and N (penalty scorefor mismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when the cumulative alignment scorefalls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

[0079] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

[0080] To obtain gapped alignments for comparison purposes, Gapped BLAST(in BLAST 2.0) can be utilized as described in Altschul et al., NucleicAcids Res. 25:3389 (1997). Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al., supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of100, M=5, N=−4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a wordlength (W) of 3, anexpectation (E) of 10, and the BLOSUM62 scoring matrix. See the worldwide web at ncbi.nlm.nih.gov. Alignment may also be performed manuallyby visual inspection.

[0081] For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

[0082] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences makes reference toa specified percentage of residues in the two sequences that are thesame when aligned for maximum correspondence over a specified comparisonwindow, as measured by sequence comparison algorithms or by visualinspection. When percentage of sequence identity is used in reference toproteins it is recognized that residue positions which are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

[0083] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

[0084] (e)(i) The term “substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%,93%, or 94%, and at least 95%, 96%, 97%, 98%, or 99% sequence identity,compared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 70%, at least 80%, 90%, at least 95%.

[0085] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions (see below). Generally, stringent conditions are selected tobe about 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

[0086] (e)(ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%,96%, 97%, 98% or 99%, sequence identity to the reference sequence over aspecified comparison window. Optimal alignment is conducted using thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.48:443 (1970). An indication that two peptide sequences aresubstantially identical is that one peptide is immunologically reactivewith antibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution.

[0087] For sequence comparison, typically one sequence acts as areference sequence to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0088] As noted above, another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions. The phrase“hybridizing specifically to” refers to the binding, duplexing, orhybridizing of a molecule only to a particular nucleotide sequence understringent conditions when that sequence is present in a complex mixture(e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers tocomplementary hybridization between a probe nucleic acid and a targetnucleic acid and embraces minor mismatches that can be accommodated byreducing the stringency of the hybridization media to achieve thedesired detection of the target nucleic acid sequence.

[0089] “Stringent hybridization conditions” and “stringent hybridizationwash conditions” in the context of nucleic acid hybridizationexperiments such as Southern and Northern hybridizations are sequencedependent, and are different under different environmental parameters.Longer sequences hybridize specifically at higher temperatures. Thethermal melting point (T_(m)) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl, Anal.Biochem., 138:267 (1984); T_(m) 81.5° C.+16.6 (log M) +0.41 (% GC) −0.61(% form) −500/L; where M is the molarity of monovalent cations, % GC isthe percentage of guanosine and cytosine nucleotides in the DNA, % formis the percentage of formamide in the hybridization solution, and L isthe length of the hybrid in base pairs. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the T_(m) for the specificsequence and its complement at a defined ionic strength and pH. However,severely stringent conditions can utilize a hybridization and/or wash at1, 2, 3, or 4° C. lower than the T_(m); moderately stringent conditionscan utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lowerthan the T_(m); low stringency conditions can utilize a hybridizationand/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the T_(m). Usingthe equation, hybridization and wash compositions, and desiredtemperature, those of ordinary skill will understand that variations inthe stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a temperatureof less than 45° C. (aqueous solution) or 32° C. (formamide solution),it is preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology Hybridization with Nucleic Acid Probes, part Ichapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays” Elsevier, N.Y. (1993). Generally, highlystringent hybridization and wash conditions are selected to be about 5 °C. lower than the T_(m) for the specific sequence at a defined ionicstrength and pH.

[0090] An example of highly stringent wash conditions is 0.15 M NaCl at72° C. for about 15 minutes. An example of stringent wash conditions isa 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M, more preferably about 0.01 to 1.0 M, Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. and at least about 60° C. for long probes (e.g., >50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Nucleic acids that do not hybridize to eachother under stringent conditions are still substantially identical ifthe proteins that they encode are substantially identical. This occurs,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code.

[0091] Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent conditions forhybridization of complementary nucleic acids which have more than 100complementary residues on a filter in a Southern or Northern blot is 50%formamide, e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C., and a wash in 0.1×SSC at 60 to 65° C. Exemplary low stringencyconditions include hybridization with a buffer solution of 30 to 35%formamide, 1M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and awash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to55° C. Exemplary moderate stringency conditions include hybridization in40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to1×SSC at 55 to 60° C.

[0092] By “variant” polypeptide is intended a polypeptide derived fromthe native protein by deletion (so-called truncation) or addition of oneor more amino acids to the N-terminal and/or C-terminal end of thenative protein; deletion or addition of one or more amino acids at oneor more sites in the native protein; or substitution of one or moreamino acids at one or more sites in the native protein. Such variantsmay results form, for example, genetic polymorphism or from humanmanipulation. Methods for such manipulations are generally known in theart.

[0093] Thus, the polypeptides of the invention may be altered in variousways including amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of the polypeptides canbe prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Kunkel etal., Meth. Enzymol., 154:367 (1987); U.S. Pat. No. 4,873,192; Walker andGaastra, Techniques in Mol. Biol. (MacMillan Publishing Co. (1983), andthe references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al., Atlas of ProteinSequence and Structure (Natl. Biomed. Res. Found. 1978). Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, are preferred.

[0094] Thus, the genes and nucleotide sequences of the invention includeboth the naturally occurring sequences as well as mutant forms.Likewise, the polypeptides of the invention encompass both naturallyoccurring proteins as well as variations and modified forms thereof.Such variants will continue to possess the desired activity. Thedeletions, insertions, and substitutions of the polypeptide sequenceencompassed herein are not expected to produce radical changes in thecharacteristics of the polypeptide. However, when it is difficult topredict the exact effect of the substitution, deletion, or insertion inadvance of doing so, one skilled in the art will appreciate that theeffect will be evaluated by routine screening assays.

[0095] Individual substitutions deletions or additions that alter, addor delete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence are also “conservatively modified variations.”

[0096] The term “transformation” refers to the transfer of a nucleicacid fragment into the genome of a host cell, resulting in geneticallystable inheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.

[0097] “Transformed,” “transgenic,” and “recombinant” refer to a hostcell or organism into which a heterologous nucleic acid molecule hasbeen introduced. The nucleic acid molecule can be stably integrated intothe genome generally known in the art and are disclosed in Sambrook andRussell, supra. See also Innis et al., PCR Protocols, Academic Press(1995); and Gelfand, PCR Strategies, Academic Press (1995); and Innisand Gelfand, PCR Methods Manual, Academic Press (1999). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially mismatchedprimers, and the like. For example, “transformed,” “transformant,” and“transgenic” cells have been through the transformation process andcontain a foreign gene integrated into their chromosome. The term“untransformed” refers to normal cells that have not been through thetransformation process.

[0098] A “transgenic” organism is an organism having one or more cellsthat contain an expression vector.

[0099] By “portion” or “fragment”, as it relates to a nucleic acidmolecule, sequence or segment of the invention, when it is linked toother sequences for expression, is meant a sequence having at least 80nucleotides, more preferably at least 150 nucleotides, and still morepreferably at least 400 nucleotides. If not employed for expressing, a“portion” or “fragment” means at least 9, preferably 12, more preferably15, even more preferably at least 20, consecutive nucleotides, e.g.,probes and primers (oligonucleotides), corresponding to the nucleotidesequence of the nucleic acid molecules of the invention.

[0100] As used herein, the term “therapeutic agent” refers to any agentor material that has a beneficial effect on the mammalian recipient.Thus, “therapeutic agent” embraces both therapeutic and prophylacticmolecules having nucleic acid or protein components.

[0101] “Treating” as used herein refers to ameliorating at least onesymptom of, curing and/or preventing the development of a given diseaseor condition.

II. Exemplary Biofilm-genes

[0102] Attachment of planktonic (“free-floating”) bacteria to a surfacetriggers the expression of a cassette of genes, which results in the“biofilm phenotype.” These phenotypic changes, analogous to sporulationor starvation survival, occur via the induction of RNApolymerase-associated sigma factors or through sensor-regulator proteinsthat are activated on attachment. Accordingly, a biofilm-gene of theinvention is any gene associated with the biofilm-phenotype. Forexample, in NTHi, biofilm genes include LsgG and Rfe.

[0103] LsgG is a global regulator that controls the expression of anumber of bacterial processes in NTHi, including molybdenum uptake andincorporation, proteins involved in anaerobic respiration and a familyof cross-linking enzymes involved in complex carbohydrate metabolism.

[0104] NTHi Rfe is a homolog of enzymes in other bacteria that areresponsible for the addition of the first sugar to the carrier lipidupon which the carbohydrates are assembled. The gene product of Rfe is ahomolog of undecaprenyl-phosphate alpha-N-acetylglucosaminyltransferase,which is involved in the addition of the first sugar to the carrierlipid upon which the biofilm is assembled in NTHi.

III. Expression of Biofilm Genes and Biofilm Peptides of the Invention

[0105] The biofilm genes and gene products of the invention may beproduced in host cells, particularly in the cells of microbial hosts,using techniques known to the art. Host cells for expression of theinstant genes and nucleic acid molecules are microbial hosts that can befound broadly within the fungal or bacterial families and that grow overa wide range of temperature, pH values, and solvent tolerances.

[0106] Because of transcription, translation and the proteinbiosynthetic apparatus is the same irrespective of the cellularfeedstock, functional genes are expressed irrespective of carbonfeedstock used to generate cellular biomass. Large scale microbialgrowth and functional gene expression may utilize a wide range of simpleor complex carbohydrates, organic acids and alcohols, saturatedhydrocarbons such as methane or carbon dioxide in the case ofphotosynthetic or chemoautotrophic hosts. However, the functional genesmay be regulated, repressed or depressed by specific growth conditions,which may include the form and amount of nitrogen, phosphorous, sulfur,oxygen, carbon or any trace micronutrient including small inorganicions. In addition, the regulation of functional genes may be achieved bythe presence or absence of specific regulatory molecules that are addedto the culture and are not typically considered nutrient or energysources. Growth rate may also be an important regulatory factor in geneexpression. Examples of host strain s include but are not limited tofungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Candida, Hansenula, or bacterial species such as Haemophilus,Salmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces,Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes,Synechocystis, Anabaena, Thiobacillus, Methanobacterium and Klebsiella.

[0107] Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of the any of thebiofilm gene products of the instant invention. These chimeric genescould then be introduced into appropriate microorganisms viatransformation to provide expression.

[0108] Vectors or cassettes useful for the transformation of suitablehost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

[0109] Initiation control regions or promoters, which are useful todrive expression of the expression cassettes in the desired host cellare numerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRPI, URA3, LEU2, ENO, TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, ara, tet, trp, (useful for expression in Escherichia coli) aswell as the amy, apr, npr promoters and various phage promoters usefulfor expression in Bacillus.

[0110] Termination control regions may also be derived from variousgenes native to the preferred hosts. Optionally, a termination site maybe unnecessary, however, it is most preferred if included.

[0111] To confirm the presence of the biofilm gene or biofilm peptide inthe host cell, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays well known to thoseof skill in the art, such as Southern and Northern blotting, RT-PCR andPCR; “biochemical” assays, such as detecting the presence or absence ofbiofilm, e.g., O'Toole-Kolter test, or detecting the presence ofendotoxin, e.g., using a Limulus amebocyte lysate assay; by microscopicmethods, e.g., scanning electronic microscopy; immunological means,e.g., immunoprecipitations, immunoaffinity columns, ELISAs and Westernblots; by continuous flow chamber analysis, or by any other assay usefulto identify biofilm genes and/or peptides falling within the scope ofthe invention.

[0112] To detect and quantitate bacterial mRNA, RT-PCR may be employed.In this application of PCR, it is first necessary to reverse transcribeRNA into DNA, using enzymes such as reverse transcriptase, and thenthrough the use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This techniquedemonstrates the presence of an RNA species and gives information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and only demonstratethe presence or absence of an RNA species.

[0113] While Southern blotting and PCR may be used to detect thepresence of a biofilm gene DNA, they do not provide information as towhether the gene or DNA segment is being expressed. Expression may beevaluated by specifically identifying the peptide products of the DNAsequences or evaluating the phenotypic changes brought about by theexpression of the introduced DNA segment in the host cell.

IV. Diseases and Conditions Amenable to the Methods of the Invention

[0114] Bacterial biofilms have been implicated in a number of conditionsand diseases, for example, endocarditis, pneumonia in cystic fibrosis,prosthetic infections, dental caries and dental plaque, and associatedperiodontal disease. In addition, biofilm-like pseudomonal aggregatesare found in the lungs of patients with cystic fibrosis (CF) (Singh etal., Nature, 407, 762-764 (2000)). Several lines of evidence suggestthat NTHi grows as a biofilm in the human respiratory tract. Therespiratory tract has been shown to harbor multiple strains of NTHi inseveral clinical settings, e.g., cystic fibrosis (CF), chronicbronchitis (CB), chronic obstructive pulmonary disease (COPD) and otitismedia (OM). Any bacterial infection associated with the presence of abiofilm is amenable to treatment using the methods and/or vaccine of theinvention. Therefore, it will be understood that the following list isexemplary rather than exhaustive.

[0115] Otitis Media

[0116] Otitis media (OM) is the most common reason for an ill child tovisit a physician or other health care professional and is the mostcommon reason for a child in the United States to receive antibiotics orundergo a general anesthetic. The underlying pathophysiology of OM ispoorly understood although it is clear that OM results from an interplayof infectious, environmental, and host genetics factors.

[0117] NTHi is the causative agent of acute OM as established by pureculture of the organism from middle ear fluid during disease. Inaddition, nontypeable H. infiuenzae has been implicated as a cause ofotitis media with effusion, which refers to the presence of fluid in themiddle ear in the absence of acute symptoms. Although most effusionsfrom acute OM are culture-positive for bacteria (predominantlyHaemophilus influenzae, Streptococcus pneumonia, and Moraxellacatarrhalis), the majority of chronic effusions are cultural-negative,refractory to antibiotic treatment, and positive for a variety ofinflammatory mediators.

[0118] Chronic Bronchitis

[0119] NTHi has been recovered from the lower airways of stable and ofacutely ill chronic bronchitis (CB) patients, whereas it was not foundin the lower respiratory tract of healthy adults. In patients with CB,NTHi appears to be associated with recurrent and/or persistentinfections of the lower respiratory tract. NTHi appears to be able topersist in the lower respiratory tract for months and can be isolatedeven after or during antimicrobial treatment (Bandi et al., Am. J.Respir. Crit. Care Med., 164, 2114-2119 (2001).

V. Vaccines of the Invention

[0120] The present invention provides a vaccine for use to protectmammals against the colonization and/or infection associated with thepresence of biofilm-bacteria, e.g., otitis media, chronic bronchitis andthe like. In one embodiment of this invention, as is customary forvaccines, a biofilm peptide, e.g., lsgG, rfe, variants or fragmentsthereof, can be delivered to a mammal in a pharmacologically acceptablevehicle. As one skilled in the art will appreciate, it is not necessaryto use the entire gene product (i.e., peptide or protein). A selectedportion of the polypeptide (for example, a synthetic immunogenicpolypeptide corresponding to a portion of the lsgG) can be used.

[0121] As one skilled in the art will also appreciate, it is notnecessary to use a polypeptide that is identical to a native biofilmpeptide's amino acid sequence. The amino acid sequence of theimmunogenic polypeptide can correspond essentially to the correspondingnative protein's amino acid sequence. As used herein “correspondessentially to” refers to a polypeptide sequence that will elicit aprotective immunological response at least substantially equivalent tothe response generated by a native biofilm peptide. An immunologicalresponse to a composition or vaccine is the development in the host of acellular and/or antibody-mediated immune response to the polypeptide orvaccine of interest. Usually, such a response consists of the subjectproducing antibodies, B cell, helper T cells, suppressor T cells, and/orcytotoxic T cells directed specifically to an antigen or antigensincluded in the composition or vaccine of interest. Vaccines of thepresent invention can also include effective amounts of immunologicaladjuvants, known to enhance an immune response.

[0122] Alternatively, the biofilm peptide can be conjugated or linked toanother peptide or to a polysaccharide. For example, immunogenicproteins well-known in the art, also known as “carriers,” may beemployed. Useful immunogenic proteins include keyhole limpet hemocyanin(KLH), bovine serum albumin (BSA), ovalbumin, human serum albumin, humangamma globulin, chicken immunoglobulin G and bovine gamma globulin.Useful immunogenic polysaccharides include group A Streptococcipolysaccharide, C-polysaccharide from group B Streptococci, or thecapsular polysaccharide of Streptococci pnuemoniae. Alternatively,polysaccharides of other pathogens that are used as vaccines can beconjugated or linked to the biofilm peptide.

[0123] To immunize a subject, the biofilm peptide, or an immunologicallyactive fragment, variant or mutant thereof, is administeredparenterally, usually by intramuscular or subcutaneous injection in anappropriate vehicle. Other modes of administration, however, such asoral delivery or intranasal delivery, are also acceptable. Vaccineformulations will contain an effective amount of the active ingredientin a vehicle, the effective amount being readily determined by oneskilled in the art. The active ingredient may typically range from about1% to about 95% (w/w) of the composition, or even higher or lower ifappropriate. The quantity to be administered depends upon factors suchas the age, weight and physical condition of the animal or the humansubject considered for vaccination. The quantity also depends upon thecapacity of the animal's immune system to synthesize antibodies, and thedegree of protection desired. Effective dosages can be readilyestablished by one of ordinary skill in the art through routine trialsestablishing dose response curves. The subject is immunized byadministration of the biofilm peptide or fragment thereof in one or moredoses. Multiple doses may be administered as is required to maintain astate of immunity to the biofilm-producing bacterium of interest, e.g.,Haemophilus influenzae.

[0124] Intranasal formulations may include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

[0125] Oral liquid preparations may be in the form of, for example,aqueous or oily suspension, solutions, emulsions, syrups or elixirs, ormay be presented dry in tablet form or a product for reconstitution withwater or other suitable vehicle before use. Such liquid preparations maycontain conventional additives such as suspending agents, emulsifyingagents, non-aqueous vehicles (which may include edible oils), orpreservative.

[0126] To prepare a vaccine, the purified biofilm peptide, fragment,variant, or mutant thereof, can be isolated, lyophilized and stabilized.The biofilm peptide may then be adjusted to an appropriateconcentration, optionally combined with a suitable vaccine adjuvant, andpackaged for use. Suitable adjuvants include but are not limited tosurfactants, e.g., hexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N′-N-bis(2-hydroxyethyl-propane di-amine),methoxyhexadecyl-glycerol, and pluronic polyols; polanions, e.g., pyran,dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g.,muramyl dipeptide, aimethylglycine, tuftsin, oil emulsions, alum, andmixtures thereof. Other potential adjuvants include the B peptidesubunits of E. coli heat labile toxin or of the cholera toxin. McGhee,J. R., et al., “On vaccine development,” Sem. Hematol., 30:3-15 (1993).Finally, the immunogenic product may be incorporated into liposomes foruse in a vaccine formulation, or may be conjugated to proteins such askeyhole limpet hemocyanin (KLH) or human serum albumin (HSA) or otherpolymers.

[0127] The application of a biofilm peptide, subunit or mutant thereof,for vaccination of a mammal against colonization of a biofilm producingbacterium offers advantages over other vaccine candidates.

VI. Formulations of Vaccines and Methods of Administration

[0128] The vaccines of the invention may be formulated as pharmaceuticalcompositions and administered to a mammalian host, such as a humanpatient, in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

[0129] Thus, the present compounds may be systemically administered,e.g., orally, in combination with a pharmaceutically acceptable vehiclesuch as an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

[0130] The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring may beadded. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the active compound, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, theactive compound may be incorporated into sustained-release preparationsand devices.

[0131] The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts may be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

[0132] The pharmaceutical dosage forms suitable for injection orinfusion can include sterile aqueous solutions or dispersions or sterilepowders comprising the active ingredient that are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

[0133] Sterile injectable solutions are prepared by incorporating theactive compound in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

[0134] For topical administration, the present compounds may be appliedin pure form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

[0135] Useful solid carriers include finely divided solids such as talc,clay, microcrystalline cellulose, silica, alumina and the like. Usefulliquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the present compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Adjuvants such as fragrances and additionalantimicrobial agents can be added to optimize the properties for a givenuse. The resultant liquid compositions can be applied from absorbentpads, used to impregnate bandages and other dressings, or sprayed ontothe affected area using pump-type or aerosol sprayers.

[0136] Thickeners such as synthetic polymers, fatty acids, fatty acidsalts and esters, fatty alcohols, modified celluloses or modifiedmineral materials can also be employed with liquid carriers to formspreadable pastes, gels, ointments, soaps, and the like, for applicationdirectly to the skin of the user.

[0137] Examples of useful dermatological compositions that can be usedto deliver the compounds of the present invention to the skin are knownto the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157)and Wortzman (U.S. Pat. No. 4,820,508).

[0138] Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

[0139] Generally, the concentration of the compound(s) of the presentinvention in a liquid composition, such as a lotion, will be from about0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

[0140] The amount of the compound, or an active salt or derivativethereof, required for use in treatment will vary not only with theparticular salt selected but also with the route of administration, thenature of the condition being treated and the age and condition of thepatient and will be ultimately at the discretion of the attendantphysician or clinician.

[0141] In general, however, a suitable dose will be in the range of fromabout 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg ofbody weight per day, such as 3 to about 50 mg per kilogram body weightof the recipient per day, preferably in the range of 6 to 90 mg/kg/day,most preferably in the range of 15 to 60 mg/kg/day.

[0142] The compound is conveniently administered in unit dosage form;for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

[0143] Ideally, the active ingredient should be administered to achievepeak plasma concentrations of the active compound of from about 0.5 toabout 75 μM, preferably, about 1 to 50 μM, most preferably, about 2 toabout 30 μM. This may be achieved, for example, by the intravenousinjection of a 0.05 to 5% solution of the active ingredient, optionallyin saline, or orally administered as a bolus containing about 1-100 mgof the active ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s). The desired dose may conveniently be presented in asingle dose or as divided doses administered at appropriate intervals,for example, as two, three, four or more sub-doses per day. The sub-doseitself may be further divided, e.g., into a number of discrete looselyspaced administrations; such as multiple inhalations from an insufflatoror by application of a plurality of drops into the eye.

[0144] The following examples are intended to illustrate but not limitthe invention.

EXAMPLE 1

[0145] In order to study the effect of lsgG on biofilm formation, amutation was made in the lsgG (HI1693) by the deletion of 704nucleotides of this gene from base 52 to 756. A spectinomycin resistancegene was ligated into the site of this deletion and the resultingconstruct was transformed by homologous recombination into thechromosome of NTHi strain 2019 (FIG. 1). Strains containing the mutatedlsgG locus were selected on spectinomycin BHI plates. The site of mutantinsertion into the lsgG gene was confirmed by PCR of chromosomal DNA andSouthern blots. The amino acid and nucleotide for LsgG sequence is shownin FIG. 2.

EXAMPLE 2

[0146] NTHi Rfe is a homolog of enzymes in other bacteria, which areresponsible for the addition of the first sugar to the carrier lipidupon which the carbohydrates are assembled. A mutation was made in NTHi2019 rfe by the deletion of 681 nucleotides from base 339 to 1020 andreplace with a spectinomycin resistance cassette (FIG. 3). The aminoacid and nucleotide sequence for Rfe is shown in FIG. 4.

EXAMPLE 3

[0147] To confirm that lsgG is a global regulatory gene, we performedgene chip array analysis comparing the RNA obtain from the NTHi 2019lsgG spectinomycin mutant with RNA obtained from strain NTHi 2019 grownin BHI broth cultures for six hours. The results of these studiesdemonstrated that the expression of 59 genes was increased over 2 foldin the wildtype NTHi 2019 when compared to the mutant (Table 1). Geneswhose expression was regulated included genes involved in molybdateuptake and incorporation, genes involved in fumerate metabolism, ironutilization, carbohydrate biosynthesis and cross-linking and a number ofgenes involved in anaerobic respiration. This chip array data stronglysuggested that lsgG may play a role in biofilm formation since bothcarbohydrate cross-linking genes and genes involved in anaerobicrespiration were regulated. Both are important factors in biofilmformation and the ability of the bacteria to survive within the reducedoxygen environment of a biofilm.

EXAMPLE 4

[0148] A test for biofilm formation is the O'Toole—Kolter test. In thistest, bacteria are grown in BHI in wells in a 96 well microtiter dishovernight. Twenty microliters of 1% crystal violet is added to eachwell. This solution is removed and the plate is washed with PBS. Twohundred microliters of methanol are added to the well. If a biofilm ispresent, crystal violet is retained after staining. With the addition ofthe methanol, the crystal violet dissolves in the methanol and theresulting intensity of the blue color can be read in a microtiter platereader at an OD of 600 nm. FIG. 5 shows the results obtained withnontypeable H. influenzae strain 2019 and the 2019 mutants in the genesdescribed. These studies show that three wildtype NTHi strains 2019,7502 and 3198 and NTHi 2019lsgA produced biofilms. Studies with thestrain 2019lsgG, and 2019rfe are also shown. Biofilm production is shownto be reduced at least nine fold in the 2019 lsgG mutant. NTHi 2019 rfeis involved in the first step in complex bacterial carbohydratesynthesis by placing a sugar (usually a hexosamine) onto a carrierlipid. These studies demonstrated that no biofilm was made when NTHi209rfe was mutated.

EXAMPLE 5

[0149] In order to evaluate biofilm formation on a glass surface withNTHi 2019, NTHi 2019lsgG and NTHi 2109rfe, studies were performed in acontinuous flow chamber with these organisms (FIG. 6) over a six-dayexperimental period. All of the strains utilized in these experimentsexpress the green fluorescent protein that was carried on the low copynumber plasmid, pACYC184. The chambers were examines using a laserscanning confocal microscope every 24 hours over the six-day period.Evidence of biofilm formation could be found with the wildtype NTHistrain 2019 but not with either NTHi 2019lsgG or NTHi 2019rfe (FIG. 7).

EXAMPLE 6

[0150] In order to evaluate the ability of NTHi and the lsgG and rfemutants to produce biofilms on a human epithelial surface, four-day NTHiinfections were performed on primary human bronchial epithelial cellsgrown in tissue culture. The results of these studies are shown in FIG.8. As can be seen an extensive microbial biofilm extends over theprimary bronchial epithelial surface by day four in the cells infectedwith NTHI strain 2019. Studies of the NTHi 2019lsgG and NTHi 2019rfemutants showed no biofilm formation by day four (FIG. 7).

EXAMPLE 7

[0151] The abbreviations used in this example are: NTHi, non-typeableHaemophilus influenzae; CFU, colony forming units; LOS,lipooligosaccharide; SEM, scanning electron microscope; Kdo,2-keto-3-deoxy-D-manno-octulosonic acid; PEA, phosphoethanolamine;NeuAc, N-acetylneuraminic acid; CMP-NeuAc, cytidine monophosphateN-acetylneuraminic acid; PBS; phosphate buffered saline; LPS,lipopolysaccharide; PCR, polymerase chain reaction; gfp, greenfluorescent protein; kb, kilobase pair: EDTA, ethylenediaminetetraaceticacid; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbentassay; LOS,lipooligosaccharide; MALDI-MS, matrix assisted laserdesorption ionization mass spectrometry; ChoP, phosphorylcholine;GalNAc, N-acetylgalactosamine; Hex, hexose; HexNAc, N-acetylhexosamine;Hep, L-glycero-D-manno-heptose or D-glycero-D-manno-heptose; P,phosphate; TOF, time-of-flight mass analyzer; [M-H]⁻, deprotonatedmolecular ion; m/z, mass to charge ratio; FITC, fluorsceinisothiocyanate;TRITC, texas red isothiocyanate; OCT, optimal cuttingtemperature.

[0152] Previous studies have suggested that nontypeable Haemophilusinfluenzae (NTHi) strains can form biofilms during human and chinchillamiddle ear infections. Microscopic analysis of a five-day biofilm fromNTHi 2019 grown in a continuous flow chamber demonstrated a biofilm witha diffuse matrix interlaced with multiple water channels. Studiesdisclosed herein show that biofilm production is significantly decreasedin a chemically-defined medium lacking N-acetylneuraminic acid (sialicacid). Based on these observations, NTHi 2019 mutations were examined inseven genes involved in carbohydrate and lipooligosaccharidebiosynthesis. NTHi 2019 mutants in CMP-NeuAc synthetase (siaB), one ofthe three NTHi sialyltransferases (siaA), and a homolog ofundecaprenyl-phosphate α-N-acetyl-glucosaminyl-transferase (wecA)produced significantly reduced amounts of biofilm. NTHi 2019 mutationsin phosphoglucomutase (pgm), UDP-galactose-4-epimerase, and two otherNTHi sialyltransferases (lic3A and lsgB) produced biofilms equivalent toor greater than the parent strain. The NTHi 2019pgm biofilm was studiedwith the Maachia amurensis-FITC and Sambucus nigra-TRITC lectins.Sambucus nigra-TRITC lectin bound to this biofilm while Maachiaamurensis-FITC lectin did not. Sambucus nigra-TRITC lectin binding wasreversed by inhibition with αα→6 neuraminyllactose and by treatment ofthe biofilm with Vibrio cholera neuraminidase prior to incubation withthis lectin. MALDI-TOF analysis of lipooligosaccharide isolated from thebiofilm, planktonic phase, and plate-grown organisms showed mostsialylated glycoforms increase 2-fold to 4-fold when the LOS is derivedfrom planktonic or biofilm organisms. These studies indicate that NTHi2019 produces a biofilm containing αα→6 linked sialic acid and that thesialic acid content of lipooligosaccharides increases concomitantly withthe organisms transition to a biofilm form.

Introduction

[0153] Bacterial biofilms have been defined as communities of bacteriaintimately associated with each other and included within an exopolymermatrix. These biological units exhibit their own properties, which arequite different in comparison with those showed by the single species inplanktonic form (1). Numerous bacterial species are capable of producingbiofilms.

[0154] Nontypeable Haemophilus infiuenzae (NTHi) is a gram-negativecocco-bacillus that frequently colonizes the human nasopharynx. NTHi isa frequent cause of otitis media in children (2) and acute bronchitisand pneumonia in patients with chronic obstructive pulmonary disease(3). Studies in a chinchilla otitis media model infected with NTHi haveindicated that biofilms are produced as a part of this infection (4).Ehrlich et al. have shown scanning electron microscopy (SEM) andconfocal images of biofilm formation on tympanostomy tubes collectedfrom children with otitis media (4).

[0155] The purpose of this study was to identify NTHi genes involved inbiofilm biosynthesis. Using several different techniques to identifybiofilm formation, mutations in NTHi genes homologous to aundecaprenyl-phosphate α-N-acetyl-glucosaminyl-transferase (wecA) wereshown to produce little to no biofilm. In addition, a mutant in a NTHisialyltransferase (siaa) and a mutant in CMP-NeuAc synthetase (siaB)resulted in significantly reduced biofilm production. Lectin studies andenzymatic analysis suggested that the sialic acid (N-5-acetylneuraminicacid, NeuAc) was a terminal sugar attached to a N-acetyl-hexosamine(probably N-acetyl-galactosamine) in an αα-6 linkage. A number ofsialylated lipooligosaccharide (LOS) glycoforms increased during thebiofilm and planktonic growth.

Experimental Procedures

[0156] Bacteria and culture conditions: The bacterial strains used inthis study are described in Table 1. TABLE 1 Bacterial Strains andVectors Source Strain or Plasmid Genotype or Reference pGBgfp::cat gfpin pGB2 Herein (6) NTHi 2019 non-typeable Haemophilus Ref. (9)influenzae NTHi 2019:gfp gfp-expressing NTHi 2019 Herein NTHi 2019 galEUDP-galactose-4-epimerase Herein (51) NTHi 2019 lic3A sialyltransferase(47,52) NTHi 2019 lsgB sialyltransferase (47) NTHi 2019 pgmphosphoglucomutase (53) NTHi 2019 pgm::gfp gfp-expressing HTHI 2019pgmHerein NTHi 2019 wecA undecaprenyl-phosphate α-N- Hereinacetylglucosaminyltransferase NTHi 2019 wecA::gfp gfp-expresing NTHi2019wecA Herein NTHi 2019 siaA sialyltransferase (47) NTHi 2019 siaBCMP-NeuAc synthetase (29) NTHi 2019 siaB::gfp gfp-expressing NTHi2019siaB Herein NTHi 2019 nanA N-acetylneuraminate lysase NTHi 2019nanA::pgm N-acetylneuraminate lysase, This study Phosphosglucomutase

[0157] NTHi strain 2019 is a clinical isolate from a patient withchronic obstructive pulmonary disease (5). This strain was reconstitutedfrom a frozen stock culture and propagated on brain heart infusion (BHI)agar (Difco, Detroit, Mich.) supplemented with 10 μg/ml hemin (SigmaChemical Co., St. Louis, Mo.) and 10 μg/ml nicotinamide adeninedinucleotide (NAD, Sigma) at 37° C., 5% CO₂.

[0158] Construction of gfp expressing NTHi: NTHi strains weretransformed with the plasmid pGB2:cat (6) expressing green fluorescentprotein (gfp) using a modification of the method of Williams et al. (7).

[0159] Construction of NTHi 2019galE: Previous studies have shown thatgalactose-4-epimerase is encoded by gene HI0351 in the Institute forGenomics database (8). Using the upstream primer 5′GCTGGTTATATCGGTTCT3′(SEQ ID NO:5) and the downstream primer 5′ GATCAGAATAGCAAGTCGC3′ (SEQ IDNO:6), a 882 bp fragment of DNA was amplified from NTHi 2019 chromsomalDNA and ligated into pCR2.1. This fragment was sequenced and shown tocontain almost the entire HI0351 (galE). A unique Eco47RIII site at bp737 was identifed in galE. The gene was digested, and anerythromycin-resistance cassette was cloned into this site. The mutationwas confirmed by restriction digestions. The plasmid containing themutated galE gene was linearized and transformed into NTHi 2019 aspreviously described (9). The mutation was confirmed by PCR and SouthernBlot analyses of the chromosomal NTHi 2019 galE DNA.

[0160] Construction of wecA::spec deletion mutant: To construct adeletion mutation in NTHi 2019 wecA (HI1716), a NTHi 2019 phagmidgenomic library was screened with a wecA digitoninin-labeled probe.Positive plaques were purifed, and excision was performed. The plasmidpBK-CMVHIA2wecA was isolated, and sequencing verified the presence ofHI1714, HI1715, wecA (HI1716), and 912 bp of the 3′ end of HI1719 in a3449 bp insert. The gene order in strain 2019 is different than H.influenzae RDKW20, and open reading frames HI1718 and HI1719 are notadjacent to HI1716. This fragment was moved into pGEM3Zf(+) at the SacIand SamI sites. The plasmid was restricted with BclI and NsiI. Thisremoved 682 bp of sequence internal to wecA. A spectinomycin-resistance(spec) cassette was cloned into the BclI and NsiI sites. This constructwas verified by DNA sequencing and diagnostic DNA restriction enzymedigests. The mutation was introduced into NTHi 2019 by linearizing theplasmid construct as above. Transformants were plated onto supplementedBHI agar containing 25 μl of spectinomycin. Confirmation of the deletionmutation in NTHi 2019 wecA was determined by PCR and Southern Blotanalyses.

[0161] Construction of nanA::kan deletion mutant: In order to study theincorporation of NeuAc into biofilm, mutations were made in theHaemophilus N-acetylneuraminate lysase gene, nanA (TIGR locus HI0142).The DNA sequence from H. influenzae Rd KW-20 was used to construct twoprimers 5′-CCTACGATATGAATAGGATCATTACG-3′ (SEQ ID NO:7) and5′-CAGTAGCTAACCCCAATACAAAAG-3′ (SEQ ID. NO:8). The polymerase chainreaction was used to amplify a 2612 bp fragment of DNA which was clonedinto pCR2.1-TOPO (InVitrogen, Carlsbad, Calif.). The fragment wasremoved by EcoRI digestion, inserted into an EcoRI digested pUC19,verifed to contain Haemophilus nanA by sequencing, and a kanamycincassette was inserted into nanA using TN::EX<KAN2> (Epicentre, Madison,Wis.) according to manufacturer's instructions. The plasmid containingthe mutated nanA gene was linearized and transformed into NTHi 2019 andNTHi 2019pgm as previously described (9). The presence of the kancassette within nanA in both mutants was confirmed by PCR and Southernblot analyses.

[0162] Biofi/m growth assay: Biofilm produced by NTHi 2019 and the NTHi2019 mutants described in Table 1 was analyzed using the microtiterplate assay described by O'Toole-Kolter (10,11). This assay is referredherein as the O'Toole-Kolter assay. An overnight broth culture of eachstrain in BHI was diluted 1:200 in fresh broth. 200 μl of thesesuspensions were inoculated in quadruplicate into outside wells of a 96well tissue culture plate (Nalgene Nunc International Co., Naperville,Ill.). The plates were incubated at 37° C., 5% CO₂ for 24 hours. Beforebiofilm quantitation, growth was assessed by measuring the OD₄₉₀. Toquantitate biofilm formation, 20 μl of crystal violet (FisherScientific, Pittsburgh, Pa.) was added to each well, and plates wereincubated at room temperature for 15 minutes. Plates were then washedvigorously with distilled water and air-dried. A volume of 230 μl of 95%ethanol was added to each well, and the OD₆₀₀ was measured. All strainswere tested in quadruplicate and average biofilm formation wascalculated from three different experiments.

[0163]¹⁴C NeuAc incorporation studies: Studies were performed to compare¹⁴C NeuAc incorporation into NTHi 2019 nanA and NTHi 2019 nanA:.pgmbiofilms. The strains were grown for 24 hours in microtiter wells at 37°C. in 5% CO₂ in RPMI (Gibco, Grand Island, N.Y.) supplemented with 0.5μg/ml protoporphyrin IX, 10 μg/ml NAD, 20 μM NeuAc and 82.5 nCi/ml ¹⁴CNeuAc (55 mCi/mM, American Radiolabeled Chemicals, Inc., St. Lousi,Mo.). The medium was carefully removed from each well and the wellswashed three times with distilled water. The biofilm was harvested in100 μl of Microscint© (Packard Meridian, Conn.) and 30 μl from each wellwas counted in a TopCounter™ (Parchard, Downers Grove, Ill.).

[0164] Laser scanning confocal microscopy (LSCM) in a continuous flowchamber: To access biofilm formation, gfp-expressing NTHi were grown ina flow chamber the size of which was 5×35×1 mm, similar to thosedescribed previously (12). The biofilm medium was composed of MorsesDefined Medium (13) diluted 1:10 with PBS and supplemented with 10 μg/mlhemin,10 μg/ml NAD, and 1 μg/ml chloramphenicol (Sigma). Depending onthe experimental conditions, 20 μM NeuAc (Sigma) was added to thismedium. To infect the flow chamber, approximately 10⁸ CFU/ml in freshbiofilm medium was placed in the chamber at 37° C. for one hour. Biofilmmedium flow was then started and maintained at a constant rate of 180μl/min. For NeuAc-free experiments, protoporphyrin IX (0.5 μgm/ml) wasadded to the medium in place of hemin. Confocal images were obtainedusing a Bio-Rad scanning confocal microscope. All the microscopes usedin these studies are located at the Central Microscopy Research Facilityat the University of Iowa (Iowa City, Iowa).

[0165] Live/Dead staining of biofilm from continuous flow chamber: Forevaluation of the live and the dead bacteria present in the biofilmmatrix, NTHi 2019 was grown in a flow chamber as described above. After2 or 5 days, the flow chamber was carefully disconnected. The LIVE/DEADBacLight bacteria viability kit (Molecular Probes, Eugene, Oreg.) wasused to visualize the live and the dead bacteria within the biofilm.Briefly, SYTO 9 (Component A) and propidium iodide (Component B) weremixed in a 1:1 ratio. Three microliters of the viability stain wereadded to 1 ml of PBS. Medium in the chamber was aseptically replacedwith the stain-PBS mixture. The chamber was incubated for 15 minutes at37° C. One ml of sterile PBS was then added to the chamber to flush awayexcess stain. The chamber was immediately visualized using the ZeissConfocal microscope at 10× magnification. The images were compiled ascross-sections of a z-series.

[0166] SEM analysis of the biofilm: Biofilms were processed for scanningelectron microscopy (SEM) and viewed using the Hitachi S-4000 scanningelectron microscope (14). Briefly, coverslips were fixed in a 2% osmiumtetroxide/perfluorocarbon solution for 2 hours, dehydrated with three100% ethanol washes, and dried using a critical point dryer to preservebiofilm formation. The coverslips were then mounted onto stubs usingcolloidal silver and sputter-coated with gold palladium.

[0167] Lipooligosaccharide preparation and neuraminidase treatment: LOSwas prepared by a modification of the Hitchcock and Brown method (15).Organisms were grown on a solid BHI medium supplemented with 10 μg/mlhemin, 10 μg/ml NAD, and 20 μM NeuAc. Organisms from a single plate weresuspended in 2 ml of PBS to a final OD₆₅₀ of 0.9. Bacteria were washedtwice with PBS, resuspended in 200 μl of lysis buffer (0.06 M Tris, 10mM EDTA, 2.0% SDS, pH 6.8) and incubated in a boiling water bath for5-10 minutes. The samples were allowed to cool, and 30 μl of aproteinase K (Sigma) solution (2.5 mg/ml diluted in lysis buffer) wasadded to 150 μl of the boiled sample. The samples were incubated at 37°C. for 16-24 hours. LOS was precipitated by adding {fraction (1/10)}volume of 3 M sodium acetate and 2 volumes of 100% ethanol, put on dryice for 10 minutes or in a −80° C. freezer for 1 hour, and thencentrifuged at 15,000×g for 5 minutes. The samples were washed twicewith 70% ethanol and brought up in ddH₂O to a final volume of 180 μl andlyophilized.

[0168] O-deacylation of LOS samples: LOS (<100 μg) from planktonic andbiofilm H. influenzae strain 2019 was O-deacylated by treatment with 30μl of anhydrous hydrazine (Sigma) at 37° C. for 40 minutes, withoccasional vortexing. Samples were then cooled in an ice bath, treatedwith 5 volumes of ice-cold acetone added drop-wise, and allowed to sitat −20° C. for 1 hour. After centrifugation (12,000×g, 30 minutes, 4°C.), the supernatants were removed and the pelleted O-deacylated LOS waswashed with 100 μl of chilled acetone and centrifuged a second time(12,000×g, 30 minutes, 4° C.). Following removal of the supernatants,the pellets were dissolved in 100 μl of Milli-Q de-ionized water(Millipore, Corp., Billerica, Mass.) and centrifuged a third time(12,000×g, 30 minutes, 4° C.) to remove traces of water-insolublematerial remaining in the samples. Finally, the supernatants(water-soluble O-deacylated LOS) from this extra centrifugation wereremoved, transferred to new vessels, and evaporated to dryness. The LOSfrom plate-grown NTHi strain 2019 (0.5 mg) was O-deacylated in a similarfashion using 100 μl of anhydrous hydrazine.

[0169] Neuraminidase-treatment of O-deacylated LOS: To remove NeuAc,aliquots of the O-deacylated LOS samples (estimate<30 μg) were digestedwith immobilized neuraminidase from Clostridium perfringens type VI-A(Sigma) in 40 μl of 10 mM ammonium acetate, pH 6.0, for 21 hours at 37°C. The immobilized enzyme was pelleted by centrifugation (12,000×g, 20minutes, 4° C.) and the supernatants were removed. Pellets were washedtwice with 50 μl of buffer followed by centrifugation (12,000×g, 20minutes, 4° C.). Combined supernatants were evaporated to dryness,redissolved in 50 μl of de-ionized water, and evaporated to drynessagain.

[0170] Matrix-assisted laser desorption ionization-time of flight(MALDI-TOF) mass spectrometry: The O-deacylated LOS samples wereanalyzed by MALDI-TOF on a Voyager-DE mass spectrometer (AppliedBiosystems, Foster City, Calif.) equipped with a nitrogen laser (337nM). All spectra were recorded in the negative-ion mode using delayedextraction conditions as described in detail elsewhere (16).O-deacylated LOS was dissolved in 30 μl of de-ionized water, and 5 μlaliquots were desalted by drop dialysis on VSWP 0.025 μm pore sizenitrocellulose membranes (Millipore Corp.) over de-ionized water for 80minutes. Recovered drops were evaporated to dryness and then redissolvedin 5 μl of de-ionized water. One μl aliquots of desalted O-deacylatedLOS samples were then delivered into 0.5 ml microcentrifuge tubescontaining a small amount of cation exchange resin (Dowex 50W-X8, NH₄ ⁺form, Bio-Rad, Hercules, Calif.). Subsequently, 1 μl aliquots of matrixsolution (a saturated solution of 2,5-dihydroxybenzoic acid in acetone)were added to the samples. After brief mixing, 1 μl portions of themixture were delivered to a stainless steel MALDI target and allowed toair dry. Approximately 200 laser shots were acquired for each sample.The spectra were smoothed with a 19-point Savitsky-Golay function andmass calibrated with an external mass calibrant consisting of reninsubstrate tetradecapeptide, insulin chain B (oxidized), and bovineinsulin (all from Sigma). For comparison purposes, a two-pointcorrection was then done on the spectra using the expected fragment ionfor O-deacylated diphosophoryl lipid A (m/z 952.0) and the “B₃”glycoform of NTHi strain 2019 (m/z 2522.1). All masses are given astheir average mass values.

[0171] Lectin Analysis of Biofilms: Five-day biofilms produced by strainNTHi 2019 pgm were subjected to lectin analysis. This strain was chosenbecause it can not produce an acceptor for NeuAC on its LOS, and ourstudies had shown that it was capable of forming a biofilm in amountsequal to or greater than the parent strain. The biofilm was fixed in 4%paraformaldehyde and embedded in situ in OCT resin (Sakura Finetek USA,Inc., Torrance, Calif.) on the cover slip surface upon which it wasformed. After hardening, the cover slip was removed by freezing thesample in liquid nitrogen and shattering the glass, leaving the biofilmwithin the OCT resin. The biofilm was then cut into 1 μm thick sections.These section were studied using fluorescent microscopy with thefollowing lectins; Maachia amurensis-FITC and Sambucus nigra-TRITClectins (EY Laboratories, San Mateo, Calif.), Binding inhibitionexperiments were performed by pre-incubation of Sambucus nigra-TRITC for30 minutes with N-neuramyllactose at a concentration of 200 μg/ml.

[0172] Statistical Analysis: Statistical Analyses using paired Student'sT-test were performed using Statview for Macintosh.

Results

[0173] NTHI biofilm formation: Previous studies in the chinchilla middleear infection model and microtiter plate biofilm assay have suggestedthat NTHi can form biofilms (4). Studies with NTHi strain 2019 in theO'Toole-Kolter microtiter plate assay suggested that this strain wascapable of forming a biofilm. The ability of strain 2019 to form abiofilm was confirmed in a continuous flow chamber over 5 days of growth(FIG. 9A). A toluidine-blue stained frozen section of the NTHi 2019biofilm embedded in OCT can be seen in FIG. 9B. This shows tightlypacked matrix and organisms at the bottom of the biofilm with a morediffuse structure interlaced with water channels further from the slidesurface. Higher magnification studies using SEM are seen in FIGS.10A-10C. At the top of this structure, a pellicle formed by the biofilmmatrix can be seen (FIGS. 10A and 10B). FIG. 10C shows a lateral view ofthe channels at a higher magnification. Fibrils can be seen extendingbetween the bacteria, which may be remnants of the biofilm matrix. Usinga Live/Dead stain, it was demonstrated that at day 2, viable organismspredominated throughout the biofilm with dead organisms primarilylocalized to the glass slide surface on which the biofilm formed (FIG.11A). In contrast, by day 5, the proportion of live organisms appearedto decrease and dead organisms were seen throughout the biofilm (FIG.11B). This suggests that, in the continuous flow system, the NTHibiofilm may have a finite life span.

[0174] Analysis of LOS glycoforms: Gene expression changes in bacteriawithin biofilms (17). To more precisely examine the expression of LOSglycoforms in H. influenzae strain 2019 during growth as a biofilm, LOSwas isolated from biofilm, planktonic, and plate-grown bacteria.Isolated LOS was O-deacylated by treatment with anhydrous hydrazine andanalyzed by MALDI-TOF mass spectrometry. H. influenzae strain 2019produces a complex mixture of LOS glycoforms when grown in culturemedium (18,19). The major component of strain 2019 LOS contains alactose moiety (Galβ1→4Glcβ1→) linked to Hep^(I) of the common corestructure (Hep^(III)α1,2→Hep^(II)α1,3→Hep^(I)α1,5→Kdo(P)→lipid A)characteristic of H. influenzae LOS (18,20,21). When strain 2019 wasgrown on solid medium supplemented with NeuAc for the present study, itsLOS repertoire expanded to include new sialylated, disialylated, andpolysialylated species (FIG. 12A and Table 2). TABLE 2 List of LOSglycoforms observed in plate-grown, planktonic, and biofilm H.influenzae strain 2019. Proposed compositions Calculated [M − H]⁻ with:Glycoform NeuAc HexNAc Hex Hep Kdo(P) ChoP 1PEA 2PEA 3PEAAsialoglycoforms A 1 3 1 2113.8 2236.9 2360.0 B 2 3 1 2276.0 2399.02522.1 B_(¶) 2 3 1 1 2441.1 2564.2 2687.2 C 3 3 1 2438.1 2561.2 2684.2 D4 3 1 2600.3 2723.3 2846.4 E 5 3 1 2762.4 2885.5 3008.5 F 1 3 3 1 2641.32764.4 G 1 4 3 1 2803.5 2926.5 H 1 5 3 1 2965.6 3088.7 Sialylatedglycoforms B* 1 2 3 1 2690.3 2813.4 B** 2 2 3 1 2858.5 2981.6 3104.6 D*1 4 3 1 2891.5 3014.6 D** 2 4 3 1 3182.8 3305.8 E* 1 5 3 1 3053.7 3176.73299.8 E** 2 5 3 1 3344.9 3468.0 3591.0 F* 1 1 3 3 1 3055.6 3178.7 H* 11 5 3 1 3256.9 3379.9 3503.0 I* 1 1 6 3 1 3542.1 3665.1 I** 2 1 6 3 13710.3 3833.3 3956.4 I*** 3 1 6 3 1 4001.5 4124.6

[0175] The sialylated and disialylated forms of the major Hex2glycoform, B₃* and B₃**, contain the sialyllactose moiety observed inother strains of H. influenzae (22-26). Asialo- and sialylatedglycoforms containing HexNAc (whose proposed compositions are consistentwith structures seen in other strains of H. influenzae) (27,28) werealso more abundant in plate-grown strain 2019. Additionally, many of thehigher molecular weight sialylated glycoforms observed are consistentwith species seen in plate-grown H. influenzae type b strain A2 (9).

[0176] When compared to the LOS from plate-grown strain 2019, the LOSfrom both the planktonic and the biofilm organisms showed increasedheterogeneity (FIG. 12). One factor contributing to the increasedheterogeneity is an overall shift to lower phosphorylation states,resulting in a distribution of glycoforms containing 1, 2 or 3 PEAs foreach species. In addition to this trend, there is enhanced production ofhigher molecular weight and sialylated glycoforms in the LOS fromplanktonic and biofilm organisms, as compared to the LOS fromplate-grown strain 2019. These increases are more easily measured whenall of the phosphorylation states for a given glycoform are summed andthe results for each sample normalized (Table 3). TABLE 3 Relativeabundances of asialo- and sialylated LOS glycoforms in plate-grown,planktonic, and biofilm H. influenzae 2019. 2019 2019 2019 Summedglycoforms Plate-grown Planktonic Biofilm Asialoglycoforms A₁ + A₂ + A₃10.0 29.2 32.5 B₁ + B₂ + B₃ 100 100 100 B_(1¶) + B_(2¶) + B_(3¶) 29.2 —— C₁ + C₂ + C₃ 22.4 63.8 118.3 D₁ + D₂ + D₃ 27.7 51.5 87.8 E₁ + E₂ + E₃12.8 26.4 32.8 F₁ + F₂ — 7.6 18.2 G₁ + G₂ — 6.8 24.6 H₁ + H₂ 16.1 35.631.5 Sialylated glycoforms B₂* + B₃* 26.2 22.0 26.4 B₁** + B₂** + B₃**23.4 52.8 52.3 D₁* + D₂* 9.0 19.0 20.6 D₁** + D₂** 6.0 18.9 20.5 E₁** +E₂** + E₃** 3.8 14.6 16.0 F₂* + F₃* 14.5 19.0 20.4 H₁* + H₂* + H₃* 11.320.2 41.9 I₂* + I₃* 5.6 3.2 4.5 I₁** + I₂** + I₃** 5.3 4.2 6.1 I₁*** +I₂*** 1.7 4.4 2.6

[0177] When treated in this semi-quantitative fashion, the MALDI resultsshow increases in glycoforms ‘C—H’ in the LOS from planktonic andbiofilm organisms. These higher molecular weight glycoforms are mostabundant in the LOS derived from the biofilm organisms and many of themare acceptors for sialylation. Concomitantly, the overall level ofsialylated glycoforms is increased in LOS from planktonic and biofilmorganisms, as compared to the LOS from plate-grown strain 2019 (Table3). In a few cases, individual sialylated glycoforms remained atcomparable levels under the three growth conditions. However, mostsialylated glycoforms increase 2-fold to 4-fold when LOS is derived fromplanktonic or biofilm organisms. Such increases are seen for the doublysialylated LOS glycoforms B**, D**, and E** of planktonic and biofilmorganisms. While in most respects LOS populations from planktonic andbiofilm organisms appear quite similar, the H* glycoform appears to beexpressed most abundantly in the LOS from biofilm organisms (FIG. 12Cand Table 3).

[0178] To confirm the assignments of the sialylated glycoforms, portionsof the LOS samples from the three growth conditions were treated withimmobilized neuraminidase. The MALDI spectrum of theneuraminidase-treated LOS sample from biofilm organisms is shown in FIG.4D. In all three neuraminidase-treated LOS samples, peaks assigned assialylated glycoforms were shifted by the loss of one or more NeuAcs.

[0179] Analysis of NTHi mutants in biofilm formation: In order todetermine the role that carbohydrates might play in NTHi biofilmformation, the complex carbohydrate biosynthesis was studied in a groupof NTHi 2019 mutants. FIG. 13 shows the results of this study. SevenNTHi 2019 mutants were studied in a microtiter biofilm assay.Interestingly, the mutants, NTHi 2019 galE and NTHi 2019 pgm, formedbiofilms. This suggested that glucose, galactose, and mannose were notcomponents of the biofilm matrix. Three of the other mutants, 2019 wecA,2019 siaB, and 2019 siaA showed a significant reduction in biofilmformation in this assay. Strain 2019 wecA is a mutant in a gene withhigh homology (e⁻¹⁰¹) to undecaprenyl-phosphateα-N-acetylglucosaminyltransferase in E. coli K12. Previous studies inour laboratory (unpublished data) and studies of Hood and co-workers(29) indicate that a mutation in this gene does not affectlipooligosaccharide biosynthesis. The microtiter assay suggested thatthis transferase might be involved in the first step in biofilmbiosynthesis, that is, the addition of an initial N-acetylhexosamine tothe undecaprenol carrier lipid. Figure panel 14A and panel 14B showconfocal analysis of biofilm formation by strain 2019 gfp and 2019wecA:gfp, respectively, at day 5 in a continuous flow chamber usingdefined medium. These data confirm the microtiter assay results sinceessentially no biofilm is form in 2019 wecA while a 50 to 150 micronthick biofilm is formed with NTHi 2019. A similar study was performedwith strain 2019 siaB that showed a reduction in the height of thebiofilm to 20 to 30 microns at day 5 (FIG. 14C). The O'Toole-Kolterassay also included studies with the three strain 2019 sialyltransferasemutants, lsgB, lic3A, and siaA. A mutation in siaA resulted insignificant reduction in biofilm formation in this assay while mutationsin lsgB and lic3A did not (FIG. 13). The O'Toole-Kolter and continuousflow studies suggested that NeuAc was an important component of thebiofilm. To confirm this observation, biofilm formation was studied in acontinuous flow chamber under NeuAc limiting conditions. A chemicallydefined medium supplemented with NAD and hemin without and with 20 μMNeuAc was perfused through separate chambers infected with NTHi 2019gfp. These studies showed reduced biofilm formation in the continuousflow chamber perfused with medium without NeuAc supplementation (FIG.14D and 14E). Similar results were obtained with the biofilm microtiterassay (FIG. 15A). In order to confirm that NeuAc was incorporated intobiofilm produced by NTHi 2019 pgm, ¹⁴C NeuAc uptake studies wereperformed. These studies were performed with strains NTHi 2019 nanA andNTHi 2019 nanA: pgm. H. influenzae cannot synthesize sialic acid but itcan degrade sialic acid to N-acetylmannosamine by the action ofN-acetylneuraminate lysase (NanA). FIG. 15B shows that NTHi 2019nanA::pgm incorporates ¹⁴C NeuAc into biofilm as efficiently as NTHi2019 nanA does into biofilm and LOS.

[0180] Analysis of biofilm composition by lectin binding studies: Inorder to confirm that NeuAc was a component of the biofilm, OCT embeddedstrain 2019 pgm biofilm was studied with the Maachia amurensis andSambucus nigra lectins conjugated to fluorescein and texas redisothiocyanate, respectively. Maachia amurensis lectin bindspreferentially to a terminal NeuAc α2→3Gal, and Sambucus nigra lectinbinds preferentially to terminal NeuAc α2→6Gal. To avoid possiblebinding of these lectins to the NeuAc on NTHi LOS 2019 pgm biofilm wasstudied in these experiments. This mutant makes an LOS that is severelytruncated and lacks acceptors for NeuAc. Thus, any NeuAc detected instudies of 2019 pgm biofilm with these lectins would be present only inthe biofilm matrix. The 2019 pgm biofilm was collected after 5 days ofgrowth in the continuous flow chamber and embedded in OCT. FIG. 16A and16B shows the results of microscopic analysis of staining with theselectins before and after treatment of the biofilm with sialidase.Sambucus nigra-TRITC bound strongly to the biofilm while Maachiaamurensis-FITC gave a much less intense signal. After sialidasetreatment, Sambucus nigra lectin no longer bound to the biofilm. Inaddition, Sambucus nigra-TRITC binding to the biofilm could be inhibitedby preincubation with α2→6 N-acetylneuramyl-lactose (data not shown).There was no change in binding with Maachia amurensis-FITC aftersialidase treatment, suggesting that the binding of this lectin was notto sialic acid and that the Maachia amurensis-FITC binding was eithernon-specific or to another component of the biofilm. These studiessuggest that NeuAc is present in the 2019 pgm biofilm in an α2→6linkage.

Discussion

[0181] Biofilms are complex communities of microorganisms that developon surfaces in diverse environments (30). They are found in manydiffering environments including industrial pipelines, ventilationsystems, catheters, and medical implants. They are involved in diseasein both humans and animals. Biofilms are dynamic structures, which startby the attachment of bacteria to a surface, development ofmicrocolonies, followed by the development of the mature, structurallycomplex biofilm (30). Bacteria eventually detach from the mature andenter the surrounding fluid phase, becoming planktonic organisms thatcan then repeat the process on other parts of the surface.

[0182] Mechanisms involved in the initial attachment differ amongmicroorganisms. The initiation of a biofilm can occur in one of threeways. The first is by the redistribution of attached cells by surfacemotility. O'Toole and Kolter (31) demonstrate that the type IV pili ofPseudomonas aeruginosa play an important role in surface adherence. Thesecond mechanism in which biofilm formation can occur is from the binarydivision of attached cells (32). The third and final mechanism is therecruitment of bacterial cells from the surrounding media (33).

[0183] Once initial attachment has been made, the cells must convertfrom reversible attachment to irreversible attachment, in which thecells switch from a weak interaction with the substratum to a permanentbonding through extracellular polymers. In addition to the formation ofthe exopolymers, the bacteria form channels and pores and redistributeaway from the substratum (34).

[0184] The maintenance of a biofilm is attributed to the development andmaintenance of the exopolysaccharide matix (35). More than 300 proteinscan be detected in bacteria from mature biofilms and not in planktonicbacteria (36). These proteins fall into the classes of metabolism,phospholipid and LPS-biosynthesis, membrane transport and secretion, andadaptation and protective mechanisms. In addition, biofilm bacteria areconsidered to be in the stationary-phase partly due to the accumulationof acylhomoserine lactone within clusters (37).

[0185] Detachment is a physiologically regulated event in which bacteriawill release from the biofilm as a planktonic organism to move on toattach to other surfaces. Many different mechanisms may contribute tothe detachment process. O'Toole and Kolter (31) demonstrate thatstarvation may lead to detachment by an unknown mechanism. Steptococcusmutans produces a surface protein-releasing enzyme that mediates therelease of cells from biofilms (38). A possible trigger for the releaseof the matrix-degrading enzyme could be cell density. In addition, thepresence of homoserine lactones may cause the reduction of biofilm, asdemonstrated with Rhodobacter sphaeroides (31,39).

[0186] In P. aeruginosa, flagella and type IV pili-mediatedtwitching-motility play important roles in surface aggregation (31). InE. coli, flagella, type I pili, and curli fimbrae have been implicatedin biofilm formation (40). Motility is not absolutely necessary as manynon-motile bacteria such as Staphylococcus epidermidis and S. mutans canalso form biofilms. The microbes within the biofilm undergo changes ingene expression when compared to plate-grown or planktonic bacteria(17). It is demonstrated that Pseudomonas putida undergoes phenotypicchanges in protein expression such that different stages of biofilmdevelopment can be recognized (36).

[0187] Recent studies provide evidence that H. influenzae can produce abiofilm during otitis media in humans and in the chinchilla middle earduring experimental otitis media. Murphy and Kickham (10) demonstratethat H. influenzae pili may play a role during growth in theO'Toole-Kolter microtiter plate assay. NTHi 2019 can form a biofilm inthe O'Toole-Kolter assay as well as in a continuous flow system. Asdisclosed herein, these systems were used to identify genes involved inthe formation of the extracellular polymeric substances (EPS) of theNTHi 2019 biofilm. These studies have shown that undecaprenol, 2019siaB, and 2019 siaA are involved in the formation of the NTHi 2019 EPS.

[0188] NTHi 2019 wecA has high homology to the same gene in E. coli,Yersinia pestis, and Salmonella typhimurium (e value<e⁻¹⁰⁰). Previousstudies indicate that it plays no role in H. influenzae LOS biosynthesis(29,41,42). This gene encodes for undecaprenyl-phosphateα-N-acetyl-glucosaminyl-transferase, and homologs are shown to beinvolved in the initial step in enterobacterial common antigen (41-43)and O-antigen biosynthesis in Salmonella enterica serovar Borreze (44)and P. aeruginosa (45). S. mutans rgpG, which has homology to E. coliwecA, is involved in the biosynthesis of an extracellular polysaccharide(46). We have demonstrated in the O'Toole-Kolter assay and in acontinuous flow chamber using NTHi 2019 wecA::gfp that no biofilm isproduced by this mutant. This suggests that NTHi wecA is involved in theinitial step in biosynthesis of the biofilm and that the biofilm issynthesized on undecaprenol pyrophosphate.

[0189] NTHi SiaB is a CMP-NeuAc synthetase (47), and NTHi SiaA is asialytranferase. Mutation of either NTHi 2019 siaB or NTHi 2019 siaA(47) resulted in significantly reduced biofilm production in both theO'Toole-Kolter assay and in continuous flow chambers. Compared to NTHi2019 weca, in which no biofilm formed by five days, a small butdetectable biofilm could be seen in the continuous flow chamber withthese mutants. NTHi has two other sialyltransferase, Lic3A and LsgB(23,47), and mutations in neither of these sialyltransferases alteredNTHi 2019 biofilm formation. When H. influenzae lsgB and lic3A aremutated, SiaA can sialylate H. influenzae LOS; however, its primary rolemost probably is involvement in biofilm formation (9).

[0190] The NTHi 2019 pgm mutant can make a biofilm equivalent to orgreater than the parent strain. This would indicate that glucose andgalactose are probably not a component of the NTHi biofilm, as this geneencodes for an enzyme essential to the biosynthesis of nucleotidederivatives of these sugars. The formation of a NeuAc containing biofilmby NTHi 2019 pgm indicates that the terminal acceptor for the NeuAc ismost likely a hexosamine, most probably N-acetylgalactosamine.

[0191] The binding of Macchia ameurensis and Sambucus nigra lectins, tothe NTHi 2019pgm biofilm was studied. In these experiments, the biofilmproduced by this mutant was used because it does not produce an LOS withan acceptor for sialylation. This allowed the study of NeuAc expressionon the biofilm alone. Studies using the Sambucus nigra lectin, beforeand after V. cholera neuraminidase treatment, gave further evidence thatNeuAc is the terminal sugar in the biofilm. Macchia ameurensis lectinbinds preferentially to NeuAc in an α(2-3) linkage (48) while Sambucanigrans lectin binds preferentially to NeuAc in an α(2-6) linkage (49).The binding of Sambucus nigra lectin combined with the failure ofMacchia ameurensis lectin to bind to the NTHi 2019 pgm biofilm suggestedthat the NeuAc is incorporated via an α(2-6) linkage.

[0192] NTHi LOS undergo significant changes from the plate groworganisms to both the biofilm and planktonic bacteria. The biofilm andplanktonic LOS becomes more heterogeneous. Sialylated, disialylated, andpolysialylated species can be found. In general, most of the sialylatedglycoforms increased two- to four-fold in LOS isolated from biofilm orplanktonic organisms. This is especially true of the double sialylatedglycoforns. There is also a shift to a lower phosphorylation state inthe biofilm and planktonic LOS. Specific differences also existedbetween biofilm and planktonic LOS with the abundance of specificglycoforms increased in each.

[0193] NTHi cannot synthesize NeuAc and obtains it from its environment.Recent studies in a chinchilla middle ear infection have suggested thatNeuAc incorporation into LOS is necessary for pathogenicity (50). Theseadaptations would enhance survival within the host environment.

Bibiliography:

[0194] 1. Miller, M. B., and Bassler, B. L. (2001) Annu Rev Microbiol55, 165-199

[0195] 2. Bluestone, C. D. (1982) N Engl J Med 306, 1399-1404

[0196] 3. Murphy, T. F., and Apicella, M. A. (1987) Rev. Infect. Dis. 9,1-15

[0197] 4. Ehrlich, G. D., Veeh, R., Wang, X., Costerton, J. W., Hayes,J. D., Hu, F. Z., Daigle, B. J., Ehrlich, M. D., and Post, J. C. (2002)Jama 287, 1710-1715

[0198] 5. Campagnari, A. A., Gupta, M. R., Dudas, K. C., Murphy, T. F.,and Apicella, M. A. (1987) Infect.Immun. 55, 882-887

[0199] 6. Barcak, G. J., Tomb, J.-F., Laufer, C. S., and Smith, H. O.(1989) Journal of Bacteriology 171, 2451-2457

[0200] 7. Williams, P., Hung, W. L., and Redfield, R. J. (1996) FEMSMicrobiol Lett 137, 183-187

[0201] 8. Fleischmann, R. D., Adams, M. D., White, O., Clayton, R. A.,Kirkness, E. F., Kerlavage, A. R., Bult, C. J., Tomb, J.-F., Dougherty,B. A., Merrick, J. M., McKenney, K., Sutton, G., FitzHugh, W., Fields,C., Gocayne, J. D., Scott, J., Shirley, R., Liu, L.-I., Glodek, A.,Kelley, J. M., Weidman, J. F., Phillips, C. A., Spriggs, T., Hedblom,E., Cotton, M. D., Utterback, T. R., Hanna, M. C., Nguyen, D. T.,Saudek, D. M., Brandon, R. C., Fine, L. D., Fritchman, J. L., Fuhrmann,J. L., Geoghagen, N. S. M., Gnehm, C. L., McDonald, L. A., Small, K. V.,Fraser, C. M., Smith, H. O., and Venter, J. C. (1995) Science 269,496-512

[0202] 9. Jones, P. A., Samuels, N. M., Phillips, N. J., Munson, R. S.,Jr., Bozue, J. A., Arseneau, J. A., Nichols, W. A., Zaleski, A., Gibson,B. W., and Apicella, M. A. (2002) J Biol Chem 277, 14598-14611

[0203] 10. Murphy, T. F., and Kirkham, C. (2002) BMC Microbiol 2, 7

[0204] 11. O'Toole, G. A., and Kolter, R. (1998) Mol Microbiol 28,449-461

[0205] 12. Singh, P. K., Parsek, M. R., Greenberg, E. P., and Welsh, M.J. (2002) Nature 417, 552-555

[0206] 13. Morse, S. A., Mintz, C. S., Sarafian, S. K., Barenstein, L.,Bertram, B., and Apicella, M. A. (1983) Infect.Immun. 41, 74-82

[0207] 14. Edwards, J. L., Shao, J. Q., Ault, K. A., and Apicella, M. A.(2000) Infect Immun 68, 5354-5363.

[0208] 15. Hitchcock, P. J., and Brown, T. M. (1983) J. Bacteriol. 154,269-277

[0209] 16. Gibson, B. W., Engstrom, J. J., John, C. M., Hines, W., andFalick, A. M. (1997) J. Am. Soc. Mass Spectrom. 8, 645-658

[0210] 17. Whiteley, M., Bangera, M. G., Bumgamer, R. E., Parsek, M. R.,Teitzel, G. M., Lory, S., and Greenberg, E. P. (2001) Nature 413,860-864

[0211] 18. Phillips, N. J., Apicella, M. A., Griffiss, J. M., andGibson, B. W. (1992) Biochemistry 31, 4515-4526

[0212] 19. Gaucher, S. P., Cancilla, M. T., Phillips, N. J., Gibson, B.W., and Leary, J. A. (2000) Biochemistry 39, 12406-12414

[0213] 20. Schweda, E. K., Hegedus, 0. E., Borrelli, S., Lindberg, A.A., Weiser, J. N., Maskell, D. J., and Moxon, E. R. (1993) Carbohydr Res246, 319-330

[0214] 21. Masoud, H., Moxon, E. R., Martin, A., Krajcarski, D., andRichards, J. C. (1997) Biochemistry 36, 2091-2103

[0215] 22. Mansson, M., Hood, D. W., Li, J., Richards, J. C., Moxon, E.R., and Schweda, E. K. (2002) Eur J Biochem 269, 808-818

[0216] 23. Hood, D. W., Makepeace, K., Deadman, M. E., Rest, R. F.,Thibault, P., Martin, A., Richards, J. C., and Moxon, E. R. (1999) MolMicrobiol 33, 679-692

[0217] 24. Schweda, E. K., Li, J., Moxon, E. R., and Richards, J. C.(2002) Carbohydr Res 337, 409-420

[0218] 25. Schweda, E. K., Brisson, J. R., Alvelius, G., Martin, A.,Weiser, J. N., Hood, D. W., Moxon, E. R., and Richards, J. C. (2000) EurJ Biochem 267, 3902-3913

[0219] 26. Mansson, M., Bauer, S. H., Hood, D. W., Richards, J. C.,Moxon, E. R., and Schweda, E. K. (2001) Eur J Biochem 268, 2148-2159

[0220] 27. Cox, A. D., Hood, D. W., Martin, A., Makepeace, K. M.,Deadman, M. E., Li, J., Brisson, J. R., Moxon, E. R., and Richards, J.C. (2002) Eur J Biochem 269, 4009-4019

[0221] 28. Phillips, N. J., Apicella, M. A., Griffiss, J. M., andGibson, B. W. (1993) Biochemistry 32, 2003-2012

[0222] 29. Hood, D., Deadman, M., Allen, T., Masoud, H., Martin, A.,Brisson, J., Fleischmann, R., Venter, J., Richards, J., and Moxon, E. R.(1996) Molecular Microbiology 22, 951-965

[0223] 30. Hall-Stoodley, L., and Stoodley, P. (2002) Curr OpinBiotechnol 13, 228-233

[0224] 31. O'Toole, G. A., and Kolter, R. (1998) Mol Microbiol 30,295-304

[0225] 32. Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C.,Givskov, M., Ersboll, B. K., and Molin, S. (2000) Microbiology 146 (Pt10), 2395-2407

[0226] 33. Tolker-Nielsen, T., Brinch, U. C., Ragas, P. C., Andersen, J.B., Jacobsen, C. S., and Molin, S. (2000) J Bacteriol 182, 6482-6489

[0227] 34. Davies, D. G., and Geesey, G. G. (1995) Appl EnvironMicrobiol 61, 860-867

[0228] 35. Davies, D. G., Chakrabarty, A. M., and Geesey, G. G. (1993)Appl Environ Microbiol 59, 1181-1186

[0229] 36. Sauer, K., Camper, A. K., Ehrlich, G. D., Costerton, J. W.,and Davies, D. G. (2002) J Bacteriol 184, 1140-1154

[0230] 37. Stoodley, P., Sauer, K., Davies, D. G., and Costerton, J. W.(2002) Annu Rev Microbiol 56, 187-209

[0231] 38. Lee, S. F., Li, Y. H., and Bowden, G. H. (1996) Infect Immun64, 1035-1038

[0232] 39. Puskas, A., Greenberg, E. P., Kaplan, S., and Schaefer, A. L.(1997) J Bacteriol 179, 7530-7537

[0233] 40. Jackson, D. W., Suzuki, K., Oakford, L., Simecka, J. W.,Hart, M. E., and Romeo, T. (2002) J Bacteriol 184, 290-301

[0234] 41. Meier-Dieter, U., Barr, K., Starman, R., Hatch, L., and Rick,P. D. (1992) The Journal of Biological Chemistry 267, 746-753

[0235] 42. Meier-Dieter, U., Starman, R., Barr, K., Mayer, H., and Rick,P. D. (1990) The Journal of Biological Chemistry 265, 13490-13497

[0236] 43. Ohta, M., Ina, K., Kusuzaki, K., Kido, N., Arakawa, Y., andKato, N. (1991) Mol Microbiol 5, 1853-1862

[0237] 44. Keenleyside, W. J., Perry, M., Maclean, L., Poppe, C., andWhitfield, C. (1994) Mol Microbiol 11, 437-448

[0238] 45. Burrows, L. L., and Lam, J. S. (1999) J Bacteriol 181,973-980

[0239] 46. Shibata, Y., Yamashita, Y., Ozaki, K., Nakano, Y., and Koga,T. (2002) Infect Immun 70, 2891-2898

[0240] 47. Jones, P. A., Samuels, N. M., Phillips, N. J., Munson, R. S.,Jr., Bozue, J. A., Arseneau, J. A., Nichols, W. A., Zaleski, A., Gibson,B. W., and Apicella, M. A. (2002) J Biol Chem 277

[0241] 48. Wang, W. C., and Cummings, R. D. (1988) J Biol Chem 263,4576-4585

[0242] 49. Shibuya, N., Goldstein, I. J., Broekaert, W. F.,Nsimba-Lubaki, M., Peeters, B., and Peumans, W. J. (1987) J Biol Chem262, 1596-1601

[0243] 50. Bouchet, V., Hood, D. W., Li, J., Brisson, J. R., Randle, G.A., Martin, A., Li, Z., Goldstein, R., Schweda, E. K., Pelton, S. I.,Richards, J. C., and Moxon, E. R. (2003) Proc Natl Acad Sci USA 100,8898-8903

[0244] 51. Maskell, D. J., Szabo, M. J., Deadman, M. E., and Moxon, E.R. (1992) Mol. Microbiol. 6, 3051 -3063

[0245] 52. Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh,S., Deadman, M. E., Cody, A., Martin, A., Mansson, M., Schweda, E. K.,Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W.(2001) Mol Microbiol 39, 341-350.

[0246] 53. Swords, W. E., Buscher, B. A., Ver Steeg Ii, K., Preston, A.,Nichols, W. A., Weiser, J. N., Gibson, B. W., and Apicella, M. A. (2000)Mol Microbiol 37, 13-27.

[0247] All publications, patents and patent documents are incorporatedby reference herein, as though individually incorporated by reference.The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1 8 1 255 PRT Haemophilus influenzae 1 Met Lys Asn Thr Glu Ile Leu LeuThr Ile Lys Leu Gln Gln Ala Leu 1 5 10 15 Phe Ile Asp Pro Lys Arg ValArg Leu Leu Lys Glu Ile Gln Gln Cys 20 25 30 Gly Ser Ile Asn Gln Ala AlaLys Asn Ala Lys Val Ser Tyr Lys Ser 35 40 45 Ala Trp Asp His Leu Glu AlaMet Asn Lys Ile Ser Pro Arg Pro Leu 50 55 60 Leu Glu Arg Asn Thr Gly GlyLys Asn Gly Gly Gly Thr Ala Leu Thr 65 70 75 80 Thr Tyr Ala Glu Arg LeuLeu Gln Leu Tyr Asp Leu Leu Glu Arg Thr 85 90 95 Gln Glu His Ala Phe HisIle Leu Gln Asp Glu Ser Val Pro Leu Asp 100 105 110 Ser Leu Leu Thr AlaThr Ala Arg Phe Ser Leu Gln Ser Ser Ala Arg 115 120 125 Asn Gln Phe PheGly Arg Val Ala Gln Gln Arg Ile Ile Asp Ser Arg 130 135 140 Cys Val ValAsp Val Asn Val Gln Gly Leu Pro Thr Pro Leu Gln Val 145 150 155 160 SerIle Thr Thr Lys Ser Ser Ala Arg Leu Lys Leu Ile Thr Glu Lys 165 170 175Glu Val Met Leu Met Phe Lys Ala Pro Trp Val Lys Ile Ser Glu Gln 180 185190 Pro Leu Ala Asn Gln Pro Asn Gln Phe Pro Val Asn Ile Lys Ser Leu 195200 205 Asn Glu Glu Glu Ala Ile Leu Gln Phe Ala Glu Ser Asn Ile Glu Phe210 215 220 Cys Ala Thr Val His Gln Pro Asn Gln Trp Gln Ile Glu Gln GlnVal 225 230 235 240 Trp Ile His Ile Asp Gln Glu Gln Ile Ile Leu Ala ThrLeu Gly 245 250 255 2 765 DNA Haemophilus influenzae 2 atgaaaaacaccgaaatttt actcaccatt aaacttcaac aagcactttt tatcgatcca 60 aaacgagttcgtttactcaa agaaattcaa caatgcggtt caattaatca agctgcgaaa 120 aatgccaaagtgagctataa aagtgcgtgg gatcatttag aagccatgaa taaaatcagc 180 cctcgcccattgctggaacg aaatacaggt ggaaaaaatg gcggaggcac ggcacttact 240 acttatgccgagcgtttgct ccaactttat gatttattag aacgtacgca agaacacgcg 300 tttcatattctacaagatga atccgtaccg ttagatagtt tactcacggc aaccgcacgt 360 ttttctttacaaagtagcgc acgcaatcaa ttttttgggc gagtggcaca acaacgcatt 420 attgactctcgttgcgttgt ggatgtgaat gtgcaaggat taccaacgcc attgcaagtt 480 tctattaccacaaaaagctc ggctcgtttg aaactcatta cggaaaaaga agtgatgctg 540 atgttcaaggctccttgggt aaaaatcagt gaacaaccat tagcaaatca accgaatcag 600 ttccccgtaaatatcaaatc actcaatgaa gaggaagcca tccttcaatt tgctgaaagc 660 aacattgaattttgtgccac agtccaccag ccaaatcaat ggcaaatcga gcaacaggtt 720 tggattcacattgatcaaga gcaaattatt ttagcgacgc tgggg 765 3 355 PRT Haemophilusinfluenzae 3 Met Leu Ser Ile Phe Val Thr Phe Leu Gly Ala Phe Leu Thr LeuIle 1 5 10 15 Val Met Arg Pro Leu Ala Asn Trp Ile Gly Leu Val Asp LysPro Asn 20 25 30 Tyr Arg Lys Arg His Gln Gly Thr Ile Pro Leu Ile Gly GlyAla Ser 35 40 45 Leu Phe Val Gly Asn Leu Cys Tyr Tyr Leu Met Glu Trp AspGln Leu 50 55 60 Arg Leu Pro Tyr Leu Tyr Leu Phe Ser Ile Phe Val Leu LeuAla Ile 65 70 75 80 Gly Ile Leu Asp Asp Arg Phe Asp Ile Ser Pro Phe LeuArg Ala Gly 85 90 95 Ile Gln Ala Ile Leu Ala Ile Leu Met Ile Asp Leu GlyAsn Ile Tyr 100 105 110 Leu Asp His Leu Gly Gln Ile Leu Gly Pro Phe GlnLeu Thr Leu Gly 115 120 125 Ser Ile Gly Leu Ile Ile Thr Val Phe Ala ThrIle Ala Ile Ile Asn 130 135 140 Ala Phe Asn Met Ile Asp Gly Ile Asp GlyLeu Leu Gly Gly Leu Ser 145 150 155 160 Cys Val Ser Phe Ala Ala Ile GlyIle Leu Met Tyr Arg Asp Gly Gln 165 170 175 Met Asp Met Ala His Trp SerPhe Ala Leu Ile Val Ser Ile Leu Pro 180 185 190 Tyr Leu Met Leu Asn LeuGly Ile Pro Phe Gly Pro Lys Tyr Lys Val 195 200 205 Phe Met Gly Asp AlaGly Ser Thr Leu Ile Gly Phe Thr Ile Ile Trp 210 215 220 Ile Leu Leu LeuSer Thr Gln Gly Lys Gly His Pro Met Asn Pro Val 225 230 235 240 Thr AlaLeu Trp Ile Ile Ala Ile Pro Leu Ile Asp Met Val Ala Ile 245 250 255 IleTyr Arg Arg Val Arg Lys Gly Lys Ser Pro Phe Arg Pro Asp Arg 260 265 270Leu His Val His His Leu Met Val Arg Ala Gly Leu Thr Ser Arg Gln 275 280285 Ala Phe Leu Leu Ile Thr Phe Val Ser Ala Val Cys Ala Thr Ile Gly 290295 300 Ile Leu Gly Glu Val Tyr Tyr Val Asn Glu Trp Ala Met Phe Val Gly305 310 315 320 Phe Phe Ile Leu Phe Phe Leu Tyr Val Tyr Ser Ile Thr HisAla Trp 325 330 335 Arg Ile Thr Arg Trp Val Arg Arg Met Lys Arg Arg AlaLys Arg Leu 340 345 350 Lys Lys Ala 355 4 1065 DNA Haemophilusinfluenzae 4 atgctgagta tttttgttac ttttcttggc gcgtttttaa cattgattgtgatgcgacca 60 cttgccaatt ggattggatt agtcgataaa ccaaactatc gtaaacgtcatcaaggcaca 120 attccactaa tcggtggcgc atcgcttttt gttggtaatc tttgctattatctgatggaa 180 tgggatcaac ttcgattacc gtatctttat ttgttcagta tttttgttttattggcgatt 240 gggattttag atgatcgctt tgatattagc ccttttttaa gagcaggcattcaagcaata 300 ttggcgattt taatgatcga tcttgggaat atttatcttg atcatcttggtcaaatttta 360 gggcctttcc aattaacgct tggttcaatt ggtttgatta ttaccgtctttgccactatt 420 gcgattatta atgcctttaa tatgattgat ggtattgatg gattgcttgggggactctct 480 tgtgtttctt ttgcggcgat tggtatttta atgtatcgag atgggcaaatggatatggcg 540 cattggagtt ttgctttaat cgtatcgatt ttaccttatt tgatgctcaatctagggatt 600 ccatttggac caaaatataa ggtgtttatg ggggatgccg ggagtacattaattggcttt 660 accattattt ggattttgtt attaagtacg caaggaaaag ggcatcctatgaatccagta 720 accgcacttt ggattattgc gattcctttg attgatatgg ttgcgattatttatcgccgt 780 gtacgtaaag gtaaaagccc attccgtcca gatcgtttac atgttcatcatttaatggta 840 agagcgggtt taacatcaag acaagccttt ttattgatta cgtttgtttctgcagtttgt 900 gcaactatcg gtattttagg ggaagtttat tatgtgaatg agtgggcgatgtttgttggc 960 tttttcattt tatttttcct ttatgtctat tcaattacgc atgcatggcgaattacccgt 1020 tgggtaagaa gaatgaaacg tcgtgccaaa cggttgaaga aagca 1065 518 DNA Artificial Sequence A synthetic primer 5 gctggttata tcggttct 18 619 DNA Artificial Sequence A synthetic primer 6 gatcagaata gcaagtcgc 197 26 DNA Artificial Sequence A synthetic primer 7 cctacgatat gaataggatcattacg 26 8 24 DNA Artificial Sequence A synthetic primer 8 cagtagctaaccccaataca aaag 24

What is claimed is:
 1. A vaccine comprising an immunogenic amount of abiofilm peptide, which amount is effective to immunize a patient againsta biofilm-producing bacterial infection, in combination with aphysiologically-acceptable, non-toxic vehicle.
 2. The vaccine of claim1, wherein the biofilm peptide is a Haemophilus influenzae biofilmpeptide.
 3. The vaccine of claim 1, wherein the biofilm peptide is aLsgG gene product and/or a Rfe gene product.
 4. The vaccine of claim 3,wherein the biofilm peptide is a LsgG gene product.
 5. The vaccine ofclaim 3, wherein the biofilm peptide is a Rfe gene product.
 6. Thevaccine of claim 1, wherein the infection is caused by abacterial-biofilm.
 7. The vaccine of claim 1, wherein the infection isotitis media.
 8. The vaccine of claim 1, wherein the infection is otitismedia with effusion.
 9. The vaccine of claim 1, wherein the infection ischronic bronchitis.
 10. A method of treating or preventing a Haemophilusinfluenzae infection, comprising administering to a patient the vaccineof claim
 1. 11. A method of preventing infection or colonization ofHaemophilus influenzae in a patient by administering to the patient anagent that inhibits the production of a Haemophilus influenzae biofilmpeptide.
 12. An isolated and purified Haemophilus influenzae cellcomprising a disrupted biofilm gene of the cell, wherein the disruptionresults in a reduction of biofilm formation in the transgenicHaemophilus influenzae cell as compared to a wild-type Haemophilusinfluenzae cell.
 13. The isolated and purified Haemophilus influenzaecell of claim 12, wherein the disrupted biofilm gene is LsgG.
 14. Theisolated and purified Haemophilus influenzae cell of claim 12, whereinthe disrupted biofilm gene is Rfe.
 15. The isolated and purifiedHaemophilus influenzae cell of claim 12, wherein the biofilm gene isdisrupted by insertional inactivation.